Lecture 5 LPS structure + functions

Question 1

What is LPS?

Answer 1

common component of the outer membrane of Gram-negative bacteria
(peptidoglycan= major in positive, minor in negative)

Question 2

Primary function of LPS?

Answer 2

structural component of the outer leaflet of the OM, creates permeability barrier

Question 3

Neisseria meningitidis: characteristics?

Answer 3

• exclusively human pathogen
• 5-10%= asymptomatic carrier
  important cause of meningitis and/or sepsis
  •capsular polysaccharides are effective vaccines for serogroups A/C/W/Y but not serogroup B

Question 4

Characteristic of invasive neisseria meningitidis strains?

Answer 4

Invasive strains are always encapsulated

Question 5

What are two options when becoming infected with neisseria meningitidis?

Answer 5

• Carriage: growth at nasopharyngeal epithelial surfaces

* Disease: invasion of submucosa, blood circulation

Question 6

Why is neisseria meningitidis used as a model for LPS biosynthesis?

Answer 6

• Unique among Gram-negative bacteria: viable without LPS
• Endotoxin activity of LPS is a major disease determinant
• LPS structures show extensive antigenic variation

Question 7

What are the two parts of LPS?

Answer 7

1. Lipid A

2. LPS core oligosaccharide (directly attached to lipid A)

Question 8

Characteristics of Lipid A?

Answer 8

• membrane anchor
• Beta(1’, 6) glucosamine disaccharide
• 1, 4’ bisphosphorylated
• 4 primary acyl chains (C-14)
• 2 secondary acyl chains (C-12 & C-14)
• major determinant of endotoxin activity

Question 9

Characteristics of 2. LPS core oligosaccharide (directly attached to lipid A)?

Answer 9

• Synthesis at the inner leaflet of the inner membrane
• Consists of an inner- and an outer-core
• Inner-core structure is conserved and consists of heptose units
• Important for the stability of the outer membrane

Question 10

Many LPS biosynthesis genes display phase variation. Why?

- High frequency (10-3 to 10-4) of variants always present in bacterial population

Answer 10

“pre-adapted” for next step in the infection process
•Immune escape from bactericidal antibodies
•Switch between intracellular vs. extracellular forms
•Mucosal vs. blood growth
•Transmissive vs. invasive forms
•Binding to host cell receptors
•Serum resistance

Question 11

The outer membrane protein Imp: characteristics?

Answer 11

• partial-loss-of-function mutations in the imp gene increased OM permeability (Imp: increased membrane permeability)
• Imp =essential protein, depletion =s evere membrane defects
• imp gene = present in all Gram-negative bacteria, except those without LPS (e.g. Spirochetes)

Question 12

Does LPS reach the cell surface in the imp mutant ? How did they find out?

Answer 12

No.

Modification of LPS with sialic acid: can be removed with a certain enzyme.
Imp mutant: sialic acid cannot be removed.
Conclusion: LPS= not surface exposed.

Question 13

What inserts LPS into the outer membrane?

Answer 13

LptD-LptE complex

Energy provided: LptB molecule. LptD : LPS insertion in outer leaflet.

Question 14

LPS synthesis pathway?

Answer 14

Cytoplasm, inner leaf IM, flips to outer leaflet of IM, then transported to Lpt complex to outer leaflet of OM.

Question 15

LPS feedback inhibition: how is it done?

Answer 15

Enzyme in IM which can degrade LpxC (protein in LPS synthesis). Degradation of LpxC is normally inhibited by pbga (enzyme in I membrane).

Aggregation of LPS molecules: pbga = blocked, -> LpxC = inhibited by enzyme in IM. = no LPS biosynthesis.

Question 16

Lipid A can be modified to change the OM permeability. Modification of Lipid A acylation can be done by different enzymes:

Answer 16

PagL deacylase & Pag Pacylase: acyl composition can be rapidly modified.
lpxE & lpxF: Lipid A dephosphorylation

Question 17

PLS O-antigen: part of LPS molecule. Characteristics?

Answer 17

• Long chain, consists of repeating sugar units (up to 40) Long highly variable polysaccharide extension of repeat units
• High structural diversity
• Not found in all Gram-negative bacteria, e.g. not in mucosal bacteria
• Important for protection against complement-mediated lysis
• Used for serotyping
- can be important virulence component

Question 18

Another aspect of LPS: it is only found in the outer leaflet. System is needed to actively maintain this. Two systems are needed to actively maintain this. They are?

Answer 18

 PldA: phospholipase which can degraded phospholipids in the outer leaflet of OM
 Mla system: can pick phospholipid molecules and transport them back to the IM

Question 19

What happens upon inactivation of both systems?

Answer 19

If you inactivate both the systems the LPS deficient bacteria becomes much more viable. Shows how important these systems are.

Question 20

Why is LPS a good target?

Answer 20

• Interfering with LPS biosynthesis or export can reduce OM barrier function, making bacteria more sensitive to existing antibiotics

Question 21

How is anitibiotic resistance against LPS gained?

Answer 21

• Some antibiotics target LPS: polymyxin/colistin. Resistance mediated by modification of lipid A, adding PEA groups

Question 22

What kind of receptors recognize common microbial components?

Answer 22

TLR. Adjuvants are often molecules that trigger the TLR receptor.

Question 23

What does TLR activation result in?

Answer 23

activation of antigen-presenting cells: upregulation of costimulatory molecules, induction of proinflammatory cytokines.

Question 24

Why is is difficult to work with recognition of LPS in mice models?

Answer 24

the TLR4/MD2 recognition of LPS variants is species-specific. Difficult when e.g. working with mice, when it works in mice but not in humans.

Question 25

Lipid A mutants occur naturally among meningococcal isolates from patients. Some show reduced cytokine-inducing capacity. why can that be an advantage?

Answer 25

Could be an advantage because the bacteria stay more ‘off the radar’ this way. -> easier spreading

Question 26

Pathogenic bacteria may express underacylated LPS (lpxL1 mutation) to avoid TLR4 activation
Then, what is the advantage of the WT LPS?

Answer 26

No reduced OM barrier function.