Trypanosomiasis

Question 1

What are the three sub species of African sleeping sickness and who do they infect?

Answer 1

Trypanasoma brucei brucei (Not human infective) - Disease in livestock T. brucei gambiense (human infective - chronic) - Months/years to progress to CNS involvement. West & Central Africa T. brucei rhodiense (human infective - acute) - Can kill in weeks/months. East & Southern Africa

Question 2

Describe symptoms of ASS at the early stage

Answer 2

• Ulcer at the fly bite site - Swollen lymph nodes - Axillary (rhodiense) - Cervical (gambiense) - Fever, weakness, headache, joint pains, night sweats

Question 3

Describe symptoms of ASS when parasite develops in lymphs/blood

Answer 3

• Early stage symptoms more pronounced - Anaemia due to autoagglutination of red blood cells - Cardiovascular/endocrine/kidney disorders - Oedema - Abortion - Immune response exhaustion

Question 4

Describe advanced stage symptoms of ASS

Answer 4

• Inflammation of NS (inc. brain) - Over production of pro-inflammatory cytokines - Accumulation of lymphocytes and plasma cells at brain surface (meningo-encephalitis)

Question 5

Describe the disease model of ASS for acute and chronic diseases

Answer 5

Acute: - Early: Activated macrophages produce NO + prostaglandins, leading to lymphocyte hyperresponsiveness. (NO role confirmed through NO synthesis inhibitors and NOS KO mice

Chronic: immunosuppression associated with increased IL-10

Question 6

Briefly describe the life cycle of T. brucei

Answer 6

Mammalian host:

• Long slender form (bloodstream, proliferative, VSG coat)
• Short sumpy form (bloodstream, quiescent, no VSG)

Insect Host

• Procyclic form (mid-gut, proliferative, procyclin coat)
• Metacyclic form (quiescent, no procyclin coat)

Question 7

Describe the pattern of antigenic variation in trypanosomes

Answer 7

Series of relapsing parasitaemia events corresponding with fever peaks. Caused by changing antigens

Question 8

What is VSG and how is it expressed?

Answer 8

Variant surface glycoprotein

• GPI anchored protein (makes 10% total parasite protein, 90% of surface protein)
• Basis for evasion of immune reponse by antigenic variation
• Primary protein sequences not identical but structures very similar (most variation in antibody binding region)

Expression:

• Metacyclic (insect salivary gland stage) - limited repetoire of ~14 different VSG genes. Expression switches every 30 divisions
• Bloodstream (mammalian stage) - Can express one of ~1000 VSG genes. Switch rate is every 100+ divisions

Question 9

Describe transcription of Trypanosome VSG

Answer 9

Polycistronic transcription:

• 1 promotor driving transcription of multiple genes (5-20)
• Bidirectional (start and end characterised by histones)
• Spacing between coding regions is small
• Once transcribed, mRNA is spliced into genes

Question 10

Describe the VSG expression sites

Answer 10

• Sub-telomeric
• Expression sites define the polycistronic transcription units which contain other invarient genes - Expression Site Associtaed Genes ESAGs)
• VSG promotor is ~50 Kb upstream of VSG gene
• Transcription is drived by Polymerase 1 which exponentially upregulates transcription of VSG genes (Pol 1 usually reserved for ribosomes etc. shows importance of VSG genes)

Question 11

How are VSG genes activated

Answer 11

Homolgous recombination

Question 12

How are VSG genes not in use silenced?

Answer 12

Structural features associated with the DNA at silent VSG genes:

Base J (modified uracil) modification to DNA. Knocks polymerase 1 off DNA. Inactive VSG sites have base J through out, active only upstream

Telomeric silencing - involves telomere bindng and spreading factors (RAP1 - remove RAP1 and multiple VSG expressed)

Question 13

Describe the action of trypanosome lytic factor (TLF) in the innate immune repsonse

Answer 13

TLF a component of human serum associated with HDL.

• Two types:
  
  • TLF1 - Apol1 and HPR
  • TLF2 - Shares components of TLF1 + IGM and lipids
• Tryp must take up TLF and TLF must enter intracellular compartment for cytotoxicity to occur
• TLF endocytosed through haem receptor on tryp. Lysis rapid and irreversible, cells swell as the lysosome grows larger. influx of Cl-
• T. b.brucei killed by TLF, rhod and gamb not affected

Question 14

Describe how T.b.rhodiense is resistant to TLF

Answer 14

endocytosis blocked, TLF stays in flagellar pocket associated with SRA (serum resistance associated gene)

Question 15

Describe SRA gene

Answer 15

• Similar in structure to VSG
• Gene located in ESAG in a VSG gene expression site
• ENcodes for a truncated protein that localises to the flagellar pocket
• Important in retention of TLF
  
  • binds to Apol1

Question 16

Why is t.b.gambiense resistant to TLF?

Answer 16

Apol1 lysing inhibited by inactive TbHpHbr receptor (mutation) - doesn’t bind TLF, endocytosis blocked

Cysteine proteases target TLF 2

Also TgsGP surface glycoprotein strengthens lysosomal membrane (no bursting)

Question 17

Describe potential therapies for trypanosomiasis

Answer 17

• Truncated Apol1 engineered to remove SRA interacting domain (lytic to T.B.rhod)
• Generated immunotoxin (curative effects seen in mice)
• Delivery of baboon TLF into livestock (baboons not susceptible to T.b.rhod)