Lecture 29 - Mycobacterial Infections II

Question 1

Names for ulcer caused by M ulcerans

Answer 1

Bairnsdale ulcer

Buruli ulcer

Question 2

How was M ulcerans isolated from Bairnsdale ulcers?

Answer 2

Bacteriologists from the Alfred Hospital in Melbourne used a faulty incubator to incubate samples (couldn’t maintain 37C)

Question 3

Optimal temperature at which M ulcerans grows

Answer 3

30C

Question 4

Countries most affected by M ulcerans

Answer 4

Western and Central Aftrican nations

Mostly tropical regions

Question 5

Number of M ulcerans cases in West Africa since 2000

Answer 5

Over 10,000

Question 6

Demographic most affected by M ulcerans

Answer 6

Young children

Question 7

M ulcerans treatment

Answer 7

Responds to combination therapy
Streptomycin, rifampicin for 8 weeks
No vaccine

Question 8

M ulcernas mortality and morbidity

Answer 8

Doesn’t kill, but advanced cases can require surgery

Question 9

M ulcerans epidemiology
1)
2)
3)

Answer 9

1) Very local epidemiology
2) Transmission appears to be from environment to humans (often aquatic environment)
3) Not human-human transmission

Question 10

Point Lonsdale M ulcerans epidemic
1)
2)
3)

Answer 10

1) 3000 permanent residents
2) More than 90 cases since 2002
3) Nearby Queenscliff unaffected for a long time

Question 11

M ulcerans pathology
1)
2)
3)
4)
5)

Answer 11

1) Prominent subcutaneous necrosis
2) Dermal layer of skin remains intact, tissue below dermis becomes encrotic
3) Ulcers of skin often painless
4) Granulomas often only form when ulcer begins to heal
5) An extracellular infection

Question 12

Toxin produced by M ulcerans

Answer 12

Mycolactone

Question 13

Mycolactone
1)
2)
3)
4)

Answer 13

1) A lipid toxin
2) Small, polyketide
3) Potent immunosuppressor at low concentrations
4) Cytotoxic at higher concentrations

Question 14

Mycolactone- M ulcerans mutants

Answer 14

Avirulent

Question 15

Pre-ulcerative lesion 
1) 
2) 
3) 
4) 
5)

Answer 15

1) Small, movable nodule
2) Small bacterial load
3) Little tissue necrosis
4) Subcellular localisation
5) High local inflammatory response (IFNg)

Question 16

Ulcerative lesion
1) 
2) 
3) 
4)

Answer 16

1) High bacterial load
2) Lots of extracellular bacteria
3) Extensive tissue necrosis
4) Impaired local inflammatory response from mycolactone release

Question 17

Spontaneous healing or treatment of M ulcerans 
1) 
2) 
3) 
4)

Answer 17

1) Bacterial load decreases
2) Subcellular localisation
3) Reduced tissue necrosis
4) Local inflammatory response returns, with reduced levels of mycolactone
5) Granuloma formation

Question 18

Mycolactone structure

Answer 18

Macrolactone ring with two acyl sidechains

Question 19

M ulcerans doubling time

Answer 19

50 hours

Question 20

What is M ulcerans most closely related to?

Answer 20

M marinum (based on 16S RNA comparison)

Question 21

M ulcerans genome
1)
2)
3)
4)

Answer 21

1) 5.6MB circular genome
2) 174kb megaplasmid (pMUM001)
3) 304 copies of 2 insertion sequences
4) 771 pseudogenes

Question 22

pMUM001
1)
2)
3)

Answer 22

1) 174kb megaplasmid in M ulcerans
2) Contains genes for mycolactone synthesis
3) 81 genes
4) 3 extremely large genes

Question 23

pMUM001 very large genes
1)
2)
3)

Answer 23

1) mlsA1 (51kb)
2) mlsA2 (43kb)
3) mlsB (7kb)

Question 24

What do mlsA1, mlsA2 and mlsB encode?

Answer 24

Encode polyketide synthases (multi-enzyme complexes) that synthesise mycolactone

Question 25

``` Mycolactone polyketide synthase 1) 2) 3) 4) ```

Answer 25

1) Produce lipids by sequential addition of acetate or propionate 2) Genes are divided into modules. Each module adds an acetate or propionate onto chain 3) The more modules there are, the longer the final polyketide will be. 4) mlsA1 and mlsA2 make the macrolactone ring (spontaneous cyclisation) and one acyl chain, mlsB makes the other acyl chain. An enzyme joins the two together

Question 26

Effect of mycolactone on the immune system

Answer 26

Suppresses IL-2 secretion by T cells

Question 27

Do different M ulcerans strains make different mycolactone?

Answer 27

Yes

Question 28

Most potent mycolactone strain

Answer 28

A/B (inhibits IL-2 synthesis at the smallest concentration)

Question 29

How similar are homologous modules of mlsA1, mlsA2 and mlsB in different strains of M ulcerans?

Answer 29

Over 98% nucleotide identity between domains of the same function

Question 30

How could mycolactone polyketide synthase be used to develop drugs?

Answer 30

Substitute in different modules to produce drug of interest. | Good idea, but very hard to execute

Question 31

``` Similarities between M ulcerans, B pertussis and Y pestis 1) 2) 3) 4) 5) ```

Answer 31

1) Plague, whooping cough pathogens have many pseudogenes 2) Evidence of lateral gene transfer 3) Closely related to a non-pathogenic species 4) Many insertion sequences 5) Reduced genome compared to close, non-pathogenic relative

Question 32

Implication of similarities between M ulcerans, B pertussis and Y pestis

Answer 32

All bacteria have evolved recently to adapt to a new environment, niche

Question 33

``` Differences between M ulcerans and M marinum 1) 2) 3) 4) ```

Answer 33

M ulcerans has: 1) Reduced phospholipase C repertoire 2) Fewer glycoproteins 3) No phenolic glycolipids 4) Mycolactone-rich vesicles and cell membrane

Question 34

How were mosquitoes found to contain M ulcerans in Point Lonsdale? 1) 2) 3)

Answer 34

1) Aedes notoscriptus larvae exposed to tap water with possum faeces contamination (possum faeces contains M ulcerans) 2) Water changed, tested every instar with Taqman PCR 3) Mosquito larvae tested at each instar, and as adult, with Taqman PCR

Question 35

Results of experiment with Aedes notoscriptus larvae?

Answer 35

M ulcerans persists within mosquito larvae In midgut, mouthparts M marinum does not do this

Question 36

Animals in Point Lonsdale that act as a reservoir for M ulcerans

Answer 36

Mosquitoes, possums

Question 37

Animal reservoir in Africa for M ulcerans

Answer 37

Not found yet in mosquitoes, obvious animals