Mimm 465: Lecture 23 Staphylococci

Front 1

Describe Staphylococci characteristics

Back 1

G+
Non-motile
Non-sporing forming
Cocci
Facultative anaerobes

Front 2

What kind of morphology do they have?

Back 2

Grape-like clusters or clumps

Front 3

What is a good test to use of Staphs?

Back 3

Catalase tests
-> catalse-+

Front 4

Describe the G+ mb.

Back 4

Polysac capsule
Cell wall (thick layer of p/g): many ptns covalently linked to the p/g (some are for adhesion)
Teichoic acids:
-lipoteichoic acids: extend out of p/gl wall
-teichoic acids: directly in p/g
2-component systems: WalKR: controls cell wall synthesis, used to assemble p/g layer
GraSR: senses AMPs and regulated alanylation of teichoic acids

Front 5

What are the major staph human pathogens?

Back 5

1) S. aureus
2) S. epidermidis
3) S. saprophyticus

Front 6

Describe S. aureus

Back 6

Coagulase positive (staphylocoagulase)
Golden pigmentation on rich media

Front 7

Describe S. epidermidis

Back 7

Coagulase negative
White pigmentation of colonies
Form biofilms on plastic implants and indwelling devices
Lives on the skin

Front 8

Describe S. saprophyticus.

Back 8

Coagulase negative
Responsible for 20% UTI

Front 9

Whats the dif btw S. aureus and S. epidermidis and S. saprophyticus?

Back 9

S. epidermidis and S. saprophyticus lack most of the virulence factors found in the S. aureus genome

Front 10

What is the virulence factor responsable for blood clot formation?

Back 10

Staphylocoagulase

Front 11

Describe staphylocoagulase.

Back 11

NOT an enz
Secreted ptn that binds prothrombin in the host to form a complex: staphylothrombin

Front 12

What does thrombin do?

Back 12

Trypsin-like SER protease
-> Converts fibrinogen to fibrin leading to blood clot formation

Front 13

How is prothrombin converted to thromin?

Back 13

Proteolytic cleavage (Arg15-Ile16)
Catalyzed by factor Xa

Front 14

What happens to the protease activity of prothrombin in the presence of staphylocoagulase?

Back 14

Protease activity of prothrombin is activated without proteolytic cleavage, resulting in the conversion of fibrinogen to fibrin

Front 15

Is Staph a normal part of the microflora?

Back 15

Yes
S. aureus part of nose and throat of 30% of population
S. epidermidis is part of the skin

Front 16

What kind of pathogens are staph considered?

Back 16

Opportunistic paths

Front 17

When do staphs cause disese?

Back 17

When the staph or their exotoxins breach the epithelial layer and get to deeper tissues

Front 18

What kind of disease is S. aureus responsible for?

Back 18

Mild to fatal
-Skin infections
Osteomyelitis (chronic infection of the bones)
-Septic arthritis
-Abscesses
-Necrotizing pneumonia
-Infective endocarditis (infectcion of heart valves)
-Sepsis
-Food poisoning and toxic shock syndromes (SAgs)

Front 19

What does severity of the disease caused by S. aureus depend on?

Back 19

The strain of it
Where colonization takes place

Front 20

What is the leading cause of hospital ass't infections?

Back 20

S. aureus

Front 21

What kind of resistance does S. aureus now have?

Back 21

Methicillin
Some Vancomycin

Front 22

When did S. aureus start developing resitance to antibiotics?

Back 22

1940s: penicillin resistance

Front 23

What is common to CA-MRSA (community ass't methicillin resistant S. aureus)

Back 23

PVL positive status (almost 100% of CA-MSRA are PVL+)

Front 24

What is PVL?

Back 24

Panton-Valentine leukocidin
Pore forming toxin
Encoded on a bacteriophage integrated in the bacterial genome

Front 25

What % of S. aureus are PVL +?

Back 25

2%

Front 26

How does PVL work?

Back 26

Bacteria engulfed from neutrophil
CA-MRSA binds and produces its toxin (PVL)
Neutrophil dies
CA-MSRA escapes

Front 27

What kind of virulence factors does S. aureus produce?

Back 27

Surface ass't ptns (>20)
Secreted ptns (exotoxins)
Capsule
Siderophores
Lipoteichoic acids
Immune evasion factors
Quorum sensing

Front 28

What carries most virulence genes?

Back 28

Bacteriophages
DNA mobile elements
Plasmids

Front 29

What are Adhesins of S. aureus known as?

Back 29

MSCRAMMS
Microbial Surface Components Recognizing Adhesive Matrix Molecules

Front 30

Where do most of the MSCRAMMs bind?

Back 30

Bind ptns from the extracellular matrix

Front 31

What are adhesins for?

Back 31

Help colonize host tissues

Front 32

How are MSCRAMMs linked to the p/g cell wall?

Back 32

Covalently linked

Front 33

Describe MSCRAMMs sequence.

Back 33

C-terminal sorting signal (LPXTG motif)
Hydrophobic domain
+vely charged tail

Front 34

Page 6 MSCARMMs of S. aureus

Back 34

MSCRAMMs of S. aureus

Front 35

Which is more often ass't with implant infections (S. aureus or S. epidermidis)?

Back 35

S. epidermidis

Front 36

what is the important anchoring system for G+?

Back 36

Sortase

Front 37

Describe sortase mediated anchoring.

Back 37

Precursor is targeted to the Sec pathway by the NH2-terminal signal peptide
Sortase rec'z LPXTG motif ad cleaves the ptn btw the threonine and glycine residues
Then, sortase catalyzes the formation of an amide bond btw the surface ptn (COOH-terminal of thronine) and the NH2-terminal of the pentaglycine crossbridge

Front 38

What are sortase substrates?

Back 38

Virulence factors essential to pathogenesis
-> without them, most pathogens cannot sustain an infection

Front 39

How many Sortase enz are there?

Back 39

4
Srt A
Srt B
Srt C
Srt D

Front 40

What is Srt A for?

Back 40

Adhesion
Immune evasion
Internalization
Phage recognition

Front 41

What is sort B for?

Back 41

Iron acquisition

Front 42

What is Srt C for?

Back 42

Pili formation

Front 43

What is Srt D for?

Back 43

Spore formation

Front 44

What is the 3D structure of sortase?

Back 44

S.aureus: StrA forms a Beta barrel with 8 B strands
Cysteine 184 and Histidine 120 are essential for sortase activity and are conserved among sortase ptns

Front 45

What is a good new target for new antimicrobials?

Back 45

Sortase

Front 46

How are sortase inhibitors developed?

Back 46

mechanism based inhibition

Front 47

What cmpd was discovered using this method?

Back 47

AAEK (aryl Beta-amino(ethyl) ketone)

Front 48

How does AAEK work?

Back 48

Active site cystein of sortase forms a covalent bond with AAEK and sortase is irreversibly inactivated

Front 49

What are the different methods for iron uptake?

Back 49

1) Siderophores
2) Surface transferrin receptor (Tpm) and an iron transport system
3) Hb binding ptns and heme-iron transport system (isd genes)

Front 50

What siderophores have been identified?

Back 50

-Staphyloferrin A
-Staphyloferrin B
-Aureochelin

Front 51

What happens when S. aureus senses iron depletion conditions?

Back 51

Global iron regulator Fur, is removed from binding sites upstream of isd genes
-> leads to their expression

Front 52

Describe the isd locus.

Back 52

2 ptns with LPXTG (left side of locus): attached to p/g by SrtA
3rd gene: NPQTN: cell wall ptn, has 3 changes from the LPXTG, further down the operon, binds other Srt (SrtB)
->This gene responsible for covalently attaching IsdC to the cell wall

Front 53

What does IsdB bind?

Back 53

Hemoglobin

Front 54

What does IsdB do once it binds Hb?

Back 54

Transfers heme containing iron (HFe) to IsdA, IsdC and then to permease transport complex (IsdDEF) in the plasma mb

Front 55

What happens when heme enters the bacterial cytoplasm?

Back 55

Monoxygenase (IsdG) catalyzes the oxidative degradation of the heme to release iron

Front 56

What is iron used for?

Back 56

Basic bacterial growth

Front 57

How much of human iron is bound to Hb?

Back 57

70%

Front 58

How much of the iron pool does transferrin contain?

Back 58

7%

Front 59

How does Staph evade ROS attack?

Back 59

Catalase inactivates toxic H2O2 produced by phagocytes

Front 60

What cause golden colour of S. aureus?

Back 60

Staphloxanthin
--> Carotenoid pigment

Front 61

What is the path of staphyloxanthin close to?

Back 61

Cholesterol in humans

Front 62

What is the path of staphyloxanthin?

Back 62

Farnesyl-PP
Dehydrosqualene
4,4'-diaponeurosporene
Staphyloxanthin

Front 63

What happens if there is a deletion in CrtM?

Back 63

Can't make staphyloxanthin
.: no golden color

Front 64

What happens to crtM mutant?

Back 64

Increased susceptibility to H2O2

Front 65

What does the S. aureus pigment confer?

Back 65

Resistance to oxidant killing in vivo

Front 66

What does heterologous expression of crtMN in Strep pyogenes fo?

Back 66

Confers enhanced survival in human neutrophils
-> Makes them more resistant to H2O2

Front 67

How does the golden pigment in staphyloxanthin work?

Back 67

Protects the bacterium from phagocytic killing through its antioxidant properties

Front 68

What were tested for inhibition of CrtM?

Back 68

Human squalene synthases inhibitors (biosynth of cholesterol)

Front 69

What was BPH-652 tested to do?

Back 69

Tested in context of cholesterol lowering therapy
At [10 uM], was found to inhibit S. aureus pigment formation
Affected S. aureus survival in the kidney after intraperitoneal infection in mice

Front 70

How do quorum sensing signals work in G+ bacteria?

Back 70

Autoinducer peptides (AIPs) exported by specific transporters (AgrB)
mb-bound 2 component histidine kinase sensors (AgrC) rec'z extracellular peptides
Sensor autoP and subsequently transfers the phosphoryl gp to the cognate response regulator (AgrA)
Following P, AgrA activated/represses T of specific target genes
Page 14

Front 71

What are AIPs?

Back 71

Short peptides (7-9 residues) containing a thiolactone ring structure

Front 72

How are AIPs classified?

Back 72

In subgps cuz dif S. aureus strains and species have divergent aa seq

Front 73

How do AIP cross-inhibit between subgroups?

Back 73

Strains producing AIP-1 inhibit agr expression and virulence of strains producing other AIPs
Strains producing AIP-1 INDUCE agr expression within the same subgp

Front 74

Agr QS system

Back 74

Agr: Accessory Gene regulator
AgrB processes and exports AIP (product of agrD)
External AIP is sensed by the sensor kinase AgrC that P the response regulator AgrA, which promotes the T from P2 to P3

Front 75

What does P3 do?

Back 75

Important for virulence

Front 76

Describe how RNA III is both mRNA and a regulator

Back 76

514 nucleotidesHighly structured
5'end contains ORF encoding delta hemolysin (pore forming toxin of S.aureus)
3'end is antisens RNA that hybridizes by base pairing with target mRNA and inhibits translation of target ptns
3'end of it can also upregulate expression of other virulence genes (exoprotease, lipase, ureasem, enterotoxin, leukocidins, alpha-toxin, methicillin resistance)

Front 77

What is Ptn A involved in?

Back 77

Complement evasion

Front 78

Describe ptn A (SpA)

Back 78

5 homologous extracellular Ig binding domains in tandem (E, D, A, B, C)
Each domain can binf Fc-y
Likely prevents opsonization and phagocytosis

Front 79

What happens to S. aureus mutants that don't have ptn A?

Back 79

They are less virulent

Front 80

What are complement evasion factors?

Back 80

Capsule
Staphylokinase: activates plasminogen into plasmin, which has been shown to cleave both IgG and C3b, leading to impaired phagocytosis
Ptn A
Extracellular fibrinogen binding ptn (Efb): binds fibrinogen and C3, preventing complement activation beyond C3b attachment

Front 81

How many capsular polysac serotypes are there?

Back 81

11

Front 82

What % of infections do type 5 and 8 account for in humans?

Back 82

Type 5: 25%
Type 8: 50%

Front 83

What type are most MSRAs?

Back 83

type 5

Front 84

What do capsular polysac do?

Back 84

Prevent interaction btw C3b and complement receptor on neutrophils and .: protect bacterium from phagocytosis

Front 85

Has vaccination vs S. aureus been successful so far?

Back 85

No

Front 86

What does the StaphVax vaccine consist of?

Back 86

Capsular polysac type 5 and 8 conjugated to a carrier ptn derived from the P. aeruginosa exotoxin A

Front 87

What efficacy did StaphVax have on patients receiving renal dialysis?

Back 87

57% and its development was stopped