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Infection Biology and Microbiology > Lecture 5: LPS, periplasm, flagella > Flashcards

Lecture 5: LPS, periplasm, flagella Flashcards

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1
Q

What does LPS stand for?

A

Lipopolysaccharide

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2
Q

Describe a Lipopolysaccharide

A
  • Large complex molecule containing lipid and carbohydrate
  • Contains: lipid A, core polysaccharide, and O side chain
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3
Q

What are 2 examples of bacteria that the LPS is found in?

A

Salmonella and E.coli

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4
Q

What is the LPS called when it is free in its host cell?

A

An endotoxin

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5
Q

Is it true that LPS can induce an immune response (i.e septic shock) ?

A

Yes, and whilst this isn’t a specific function of the LPS, it does induce an immune response when it comes into contact with an innate immune system

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6
Q

do gram negative bacteria have an LPS?

A

No, only gram positive

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7
Q

What are the 3 main components of the LPS?

A

Lipid A

Core polysaccharide

O side chain

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8
Q

Describe the Lipid A component of the LPS

A
  • Made up of 2 glucosamine residues linked to fatty acids and phosphate (occasionally pyrophosphate)
  • Integrated into the outer membrane and so can act as an endotoxin and trigger an immune response
  • Straight
  • Very variable composition
  • Stabilises the outer membrane structure
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9
Q

Describe the core polysaccharide component of the LPS

A
  • Also referred to as R-antigen or R-polysaccharide
  • Has side chains of NAG, phosphate and ethanolamine

-

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10
Q

Describe the O side chain component of the LPS

A
  • Has a variable region that extends outwards, which is responsible for the antigenic makeup of bacteria. Different O side chains are linked to different diseases.
  • O side chain is flexible and bent
  • Highly variable composition (at least 20 different sugars)
  • Rough/smooth variants depend on the side chain length
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11
Q

Why do bacteria move between rough and smooth variants?

A

To protect them from the immune system

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12
Q

What are the functions of the LPS

A
  • Stability of the outer membrane (provided by Lipid A)
  • Contributes to negative charge on the surface of the bacteria (due to the core polysaccharide being charged)
  • Charged, hydrophilic external layer reduces permeability of hydrophobic substances (e.g. preventing entry of bile salts, antibiotics
    ).
  • Protects against host defences

-

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13
Q

What’s more susceptible to phagoctytosis, rough variants or smooth variants?

A

Rough variants

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14
Q

What does the loss of the O antigen in salmonella lead to ?

A

Reduced virulence

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15
Q

Describe endotoxins

A
  • Produced by many pathogens and are released during cell division or by lysis of bacterial cells, and trigger an immune response
  • Can act to prime immune system against a pathogen
  • If LPS is in the blood, can cause septic shock syndrome, of which there is no direct treatment
  • Immunogenic even in absence of living cells
  • Can be tested for using the Rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) assay
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16
Q

What are some important properties of endotoxins?

A
  • Heat stable
  • Toxic in small amounts
  • Interacts with innate immune system cells
  • Has serious infects, including: inflammation, fever, shock, and even death
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17
Q

How can endotoxins interact with innate immune system cells?

A

Can trigger the release of cytokines in a cascade

Activates transcription factors such as interferon-β and tumour necrosis factor (TNF)

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18
Q

Describe porins

A
  • Make the outer membrane more permeable than the inner membrane
  • Links the inter membrane space with the outside
  • Protein channels that facilitate the diffusion of small molecules
  • Made up of transmembrane proteins
  • Highly conserved structure
  • Form water-filled channels in the outer membrane
  • Most porins are non-specific, but some are selective
  • High thermal stability
  • Resistant to protease and detergent degradation
  • Have an unusual, stable, and closed structure

-

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19
Q

Is it true that compounds diffuse through porins and into the periplasm?

A

Yes

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20
Q

What is the periplasm?

A

the space between outer membrane and cytoplasmic membrane that has a lor of proteins

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21
Q

what does the periplasm range in size from?

A

1 to 70 nm

22
Q

is it true that the periplasm can be up to 40% of the cell volume?

A

Yes

23
Q

why does the periplasmic space have a gel like consistency?

A

Due to the abundance of proteins

24
Q

What allows for the study of the proteins and enzymes present in the periplasmic space?

A

Removal of cell walls without lysing the cells

25
Q

Describe the enzyme activity in the periplasmic space

A
  • Nutrient acquisition: Hydrolytic enzymes such as alkaline phosphatase
  • Energy conservation: e.g. electron transport proteins
  • Some peptidoglycan synthesis enzymes are periplasmic
  • Periplasmic binding proteins: Deliver specific compounds to ABC transporters in cytoplasmic membrane
  • Chemoreceptors: Involved in chemotaxis
26
Q

How can proteins be exported into the periplasm ?

A

Through the SEC pathway and through the TAT pathway (twin arginine translocase)

27
Q

Descrine the SEC pathway

A
  • Exports nascent polypeptide through cytoplasmic membrane using a translocase
  • Folding of protein occurs after translocation
  • Proteins have an N-terminal signal peptide
28
Q

Describe the TAT pathway

A
  • Exports fully folded enzymes across cytoplasmic membrane
  • Proteins have a twin arginine in N terminal region
29
Q

What 3 ways can transport occur across a cytoplasmic membrane?

A
  • Simple transport
  • Group translocation (substance is changed/phosphorylated as it crosses the membrane)
  • The ABC system (active transport)
30
Q

What are the 3 types of transporter events?

A
  • Uniporter
  • Antiporter (hydrogen)
  • Symporter
31
Q

Describe flagella

A
  • Long extracellular helical structures made of protein subunits that aid in motility
  • Up to 20 µm long (much longer than the cell)
  • connected to a motor that spins them clock- or anti-clockwise allowing bacteria to swim
  • Complex ring structures anchored into the membrane and cell wall/outer membrane
  • The flagella can act as an antigen (Different antigenic properties of shaft, tip of shaft and hook
    ).
  • The motor is driven due to transfer of protons through the ring structure (from periplasm to cytoplasm)

-

32
Q

Describe the motor of a flagella

A
  • 50 nm in diameter
  • 20 different proteins
  • Rotates at ~300 rpm (E. coli) to 1100 rpm (Vibrio alginolyticus)
33
Q

Describe the structure of flagellum filament

A
  • made of a single protein called flagellin
  • 38 – 53 kDa in most species
  • N-terminal sequence very homologous (the same) in many bacteria
  • The base is called the hook
  • The hook and the shaft have have different protein subunits
  • Single protein connecting the shaft to the motor
  • The motor moves like a rotary motor
34
Q

Is it true that the hook of the flagellum is anchored to the whole cell wall structure?

A

Yes

35
Q

Where does growth occur in a flagellum?

A

At the tip of the flagellum

36
Q

In gram negative bacteria, where is the L ring, P ring, S-M ring, and C ring located?

A

L ring in Lipopolysaccharide

P ring in Peptidoglycan

S-M ring in and on Membrane

C ring in Cytoplasm

37
Q

Describe how the flagella is synthesised

A
  • Subunits made in cytoplasm and are exported to periplasm
  • Pass up 3 nm channel in flagellum, polymerise at the tip

-

38
Q

Approximately how many flagellin subunits are there per flagellum?

A

20,000

39
Q

Why is the flagellum constantly growing?

A

to repair shear damage caused by movement

40
Q

Describe the order of steps of flagellum synthesis

A

1) MS and C rings in cytoplasmic membrane

2) Motor proteins

3) P and L ring, hook and cap

4) Flow of flagellin through hook, growth of filament

41
Q

What type of motion is the motor?

A

A rotary motion

42
Q

What powere the motor?

A
  • Protons move across the cytoplasmic membrane through the mot complex
  • This provides the energy for the motor to spin
43
Q

Does mutational analysis suggest that the motor depends on an Asp residue in Mot B? And describe this

A

Yes

  • Hypothesis: Changes in charge lead to conformational change of MotA, causing movement relative to MotB
  • Second conformational change upon loss of charge may provide another step of the motor
44
Q

Are the flagella in gram positives different to the ones in gram negatives?

A

Yes

45
Q

How are the flagella in gram positives different to the flagella in gram negatives?

A

In Gram +ve, no L and P rings

Anchor in the membrane layer is more complex

Mot proteins surround inner ring and movement of these relative to each other provides the force

46
Q

What is the H antigen?

A

Flagellum

47
Q

Is it true that Escherichia and Salmonella serotypes are defined in part by flagella structure?

A

Yes

48
Q

What is E. coli O157:H7 best known for?

A

Strain causing food-borne illness including hemorrhagic diarrhoea and kidney failure

49
Q

What is E. coli O104:H4 known for?

A

causing the 2011 Germany outbreak

50
Q

Why isn’t the H antigen of much diagnostic value?

A

Because there’s many fewer H serotypes than O serotypes (LPS O polysaccharide), better to be used in conjuction with O serotypes.

51
Q
A

Infection Biology and Microbiology (23 decks)

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