Adaptations in parasites: essential for their survival

Question 1

Why study parasitic infections?

Answer 1

-It’s a huge health problem- very common in world:
by intestinal roundworms 1/3 of the whole world is infected
Malaria. 300 mil people infected
Also others: Schisto 200 mil. and others

Question 2

Why parasites arent seen as a significant clinical problem?

Answer 2

• Not a western disease(neglected)
• Low income country diseases bring no income = so no interest in less profit
• no dual market
• sometimes completely asymptomatic/most parasites don’t cause death (except maybe malaria, one of the three killers in infectious diseases ) = so not widely researched by high-income countries either

But they cause a lot of morbidities, high DALY (how much healthy life years lost): with DALY analysis infectious diseases look much more important
Usually infectious diseases cause a lot of burden in individuals although it doesn’t kill

Question 3

Parasites vs bacteria and viruses

Answer 3

• Parasites are most complicated pathogens
• Has a large genome, up to 10-20 genes, even comparable to our genome
  eg. bacteria has less than 1 mb in comparison lol
• Has multiple stages in multiple hosts, different genes and different set up enzymes might help it to grow in different environments = other pathogens don’t have lifecycles
• They can also adapt really well for survival that drive scientific/clinical interest

Question 4

African Sleeping Sickness definition

Answer 4

Caused by Trypanosoma brucei, there’s two types on West and East that causes slightly different pathogenicity
Endemic in Subsaharan Africa, over a million people infected/depends on the control measurements of flies
Does a little zoonosis sometimes, also found on lifestock
Has flagella/movement, its extracellular unicellular protozoa, lives freely in the blood vessel with RBCs
transmitted by big and aggressive insects, tsetse fly
How do they escape immune system? = is study of interest

Question 5

Lifecycle of trypanosoma brucei

Answer 5

1) Tsetse fly takes blood meal from human/other mammals
2) trypanosome goes to fly midgut : they go procyclic and replicate
3) then trypanosome goes salivary gland and transforms to epimastigote, then metacyclic: in this form it does not divide anymore, waits until fly bites another host
4) once the parasite is given to another host: in mammal it differentiate into long slender form and replicates
5) after a while it goes short and stumpy again to be taken by a fly

Question 6

African sleeping disease phases/ symptoms

Answer 6

1st phase: blood phase, causes fever, headache, systemic inflammation, flu like
2nd phase: neurological phase, causes mental disturbance, sleep rhythm disturbances (hence sleeping sickness, people stop responding) then go into coma & death
In brain: immune surveillance after BBB is much less, so disease progresses really fast, a lot of multiplication
No effective treatment for 2nd phase: they use Arsenic wtf. xD poison, you poison yourself but they are slightly more susceptible than we are

Question 7

Interesting features of trypanosomes

Answer 7

*Clinically important pathogen: causes sleeping sickness/nagana
Nagana: affects livestock, cattle/horses. No horses are found in Subsaharan Africa because they die instantly due to Trypanosoma, also cattle are very skinny, even if there’s a lot of grass. Due to nagana: cattle produce less meat/milk, can’t be used much
*Has alternating proliferation and differentiation in the lifecycle
Now you need markers to study both proliferation and differentiation
*Has single mitochondrion per cell: which is strange. All other eukaryotes except one other protozoa has multiple mitochondria in cells
What this means: You cannot split into two randomly, you need to divide your mitochondria first! = harder to do

DNA staining: nucleus + kinetoplast (DNA of mitochondria)= a lot of DNA in mitochondria, and different from human one.
*Kinetoplasts: Their structure is also different, interconnected chains of DNA. Taking this apart to replicate is very hard, needs machinery, also would take much longer to replicate.

Why so large? = sequence, sequencing shows that what’s in the genome is not what’s encoded in the protein sequence. = also weird.
*RNA editing in mitochondria= its normally a sophisticated repair mechanism since in mitochondria often mutations happen due to ROS.
What higher eukaryotes do: they get multiple mitochondria rather than bothering, they just throw away mutated mitochondria.
You can change the mRNA however you want with this system- but requires a lot of guide RNAs (siRNA). = that’s why the genome is huge. Other organisms also use this editing strategy to make mRNA stable.

• Glycosomes
• VSG coat-antigenic variation

Question 8

Glycosomes of Trypanosomas

Answer 8

Have peroxisomal origin
-Use same import system as peroxisomes
-Has similar content: does typical peroxisome functions/biosynthesis + also degrades long chain FA
Different from peroxisome/unique features:
-Has no catalase
-Has pyrophosphate metabolism
-Allows compartmentalization of metabolism: has part of glycolysis (instead of all cytosol) and nucleotide biosynthesis

Question 9

VSG coat (explain too) + GPI anchor

Answer 9

Parasitemia during sleeping sickness fluctuate in the blood vessels, numbers keep increasing and decreasing and it continues for a really long time. = they never die completely though
Reason? VSG coat keeps changing
-Adaptive immune response recognizes, then VSG coat is changed, and not recognized by preexisting Abs anymore, then you wait for another set of Abs, the parasite multiplies, then you almost kill it again, then changes VSG: cycle goes on
VSG is encoded on telomeres, and telomeres alter, so you have a lot of combinations.
=ANTIGENIC VARIATION

Abs normally bind to trypanosomes, activate MAC + complement, create a pore, and spill out the parasite.
If VSG was a transmembrane protein: the cell would be really stiff, too much protein- protein interaction would take away its motility, would get stuck in the spleen and macrophages will kill them.
Solution: they are GPI anchored, it gives flexibility.
Discovered in trypanosomes -very abundant-, but also used widely on eukaryote proteins.

Question 10

How to switch expression of VSG genes? / gene alterations in expression

Answer 10

A/B genes, A expressed = A/B genes, express B, shut down transcription site for one, open the other: in situ switch
A/B X B/A crossover = express B instead of A now: telomere exchange
copy one gene and paste it on somewhere transcriptionally active/throw the old gene out: gene conversion
Different trypanosomes can either do one of these: all switches have another frequency of occurring
But this switch should lead to new VSGs fast enough before complete elimination + also you cannot just throw all different VSG combinations in the genome and express all at the same time= immune response will be developed to all of them = you will die.
It happens during normal telomere replication in the cell cycle, it’s not a genetic drift

Question 11

Ascaris + lifecycle

Answer 11

Giant intestinal worm, adult: 15-20 cm, extracellular obv, one of them is not that big, but they can be a lot & prevent the movement in the intestine
If you live in somewhere endemic: you can have a lot of worms until you get sick
1) Eggs are secreted from humans in feces
2) Eggs develop in moisture/and certain temperature in soil, there’s fertilized and unfertilized eggs = fertilized ones continue the transmission
There’s a shell around the eggs that protects, but it also prevents for them to get any external feeding, it uses whatever is in the shell
Also adaptations in eggshells provide hiding from immune system
3) Then you eat contaminated food and drinks, then eggs go intestine and hatch
4) Eggs develop into larvae then go to lungs, coughed and swallowed again
Swallowing the eggs back is hard: maybe can be tackled with sticky eggs
Still, very complicated inside host/needs to survive immunity/stomach acids twice/also swallowing.
Solution: no replication of worms is observed in the intestine (at the end of the cycle) if you have 1 female and 1 male: they stay that way, but they produce a lot of eggs. So production of hundreds of thousands of eggs in lifespan in a couple of years=billions! So chances of reinfection increase a lot, even 1 in a million worms that are fine.
5) After swallowing: they go stomach again. Even larvae is resistant to stomach acids
6) Then they mature into adult worms, they get together in intestine (1 male and 1 female) and sexually replicate = female produces eggs
*In order to do this very complicated lifecycle: the worm has to be not deadly (reduce virulence) , so it can survive in host and hide/and produce a lot of offspring to increase its reproduction chances evolutionarily
Need to both infect host for long and interact with host immunity = requires a lot of adaptations

Question 12

Flatworms example

Answer 12

Trematodes (flukes) example: Schistosoma Fasciola hepatica

Question 13

Helminth pylogeny

Answer 13

Worms aren’t a single phylogenic cluster
Roundworms: Nematoda - more similar to spiders/insects Antropoda, compared to flatworms, completely different morphology
Flatworms: Trematoda Cestoda
So different drugs are used: for Roundworm: Ivermectin/Albendazole
Flatworm: Praziquantel
they don’t have effect on the other class most of the times

Question 14

Fasciola hepatica + lifecycle

Answer 14

Liver fluke trematode: has an indirect cycle with 2 different hosts

1) Liver fluke is found on the liver of sheep/cattle/humans sometimes
2) It starts producing eggs, it’s released from intestine to environment
3) In water, the eggs hatch, and miracidium is released. Miracidium has cilia and it can swim.
4) It infects other snails as an intermediate host, develops in snails, and is released as cercaria.
5) Cercaria sticks to plants around, and once plants are ingested by sheep and cow, cercaria hatches after the stomach pass and is released, goes back to the liver bile duct.

Question 15

Why metabolism of Fasciola must be complicated?

Answer 15

Has distinct environments during the lifecycle:
snail/cow: no O2, there’s food -parasitic stage-
water/on grass: a lot of O2, no food -free-living stage-
=A lot of metabolism alterations are needed, which can be studied for drug targets

Question 16

Energy metabolism in mammals

Answer 16

Glycolysis + krebs + ETC = OXPHOS

Energy production if no O2: Glycolysis produces NAD: can’t put it on Krebs because it produces more NAD, no ETC if no O2.
Only chance= go for fermentation, NAD recycled but not much ATP. Can be only done for a short time in muscles for eg. lactate also accumulates, produces fast ATP using a lot of glucose in anaerobic conditions.

Question 17

metabolism in helminths / Fasciola example

Answer 17

To study: give labeled substrates+see pathway
Fasciola: free-living stage w O2: regular OXPHOS, O2 is final acceptor after ETC
No O2: complicated
-Glycolysis is not completed until pyruvate, stops in phosphoenolpyr.,
-PEP converted into malate, malate moves inside mitochondria, leading to either acetate (generates 2 NADH). Or fumarate, then succinate, then proportionate (takes 1 NAD back).
Between fumarate to succinate, the electron is donated to fumarate (oxidized), and fermentation happens.
PEP to malate conversion recycles NAD, 1 acetate (requires 2 NAD) :2 proportionate (takes 1 NAD back) to compensate redox balance

What’s different than us: additional enzymes 4 acetate formation
fumarate reductase + other ETC members in anaerobic resp. = interesting targets to study

Question 18

How did anaerobic mitochondria evolved? /fumarate reductase / transporters

Answer 18

-Earliest times: life was anaerobic: before plants, there wasn’t O2
After plants, it switched to O2 mediated mechanism
So are the adaptation in parasites now are ancient or new?

• Mitochondria can vary in size, morphology, DNA content (genome can be small or very large like Trypanosoma), protein content, ETC
• Archae just phagocytosed an alpha-proteobacteria: and it started acting as mitochondria on earliest times

Looking at the evolution of eukaryotic fumarate reductase:

• First it was free-floating in anaerobic form
• Then it got connected to the membrane with anchor, uses MQ
• Once O2 is available, it started using ubiquinone /ETC like
• In parasites now: this switched to 2nd phenotype: using RQ

Is this evolved recently/or does the evolution just go back?
-All the genes are expressed from aerobic eukaryotes, they had the O2 metabolism but decided to use this, later on, to adapt to different environments.
Transporters: ubiquitinin: aerobic
MQ mediquinin: bacteria anaerobic, their heads are very different
RQ tho what helminths use now: much more similar to ubiquitinin
So its rather a backward adaptation acquired later from helminths
A lot of different mitochondria evolved to solve O2 problem:
-All of them has fermentation metabolism even when using O2

Question 19

What’s the crucial difference in the progression of infection by parasitic protozoa vs helminth? eg. compare 1 Ascaris egg (Helminth) vs 1 trypanosoma brucei trypomastigote (Protozoa)

Answer 19

Well, 2 eggs = 2 worms only, so won’t be a heavy infection, severity depends on how many eggs you are infected with.
Helminths don’t replicate inside the host only produce eggs to infect other hosts.
(1 exception: Strongyloides sterocalis)
Also explains why helminth infections aren’t worldwide, if you stop environment contamination w stool= lifecycle cannot continue, in high income countries the cycle is broken w sanitation.
But w Trypanosoma, it multiplicates.

Question 20

Survival mechanisms of protozoa

Answer 20

Ag variation eg. Trypanosoma, Plasmodium = remains ahead of immune Ab response
Intracellular location, hidden from immune system
eg. Plasmodium, toxoplasma, leishmania
Manipulates host immune system (actually helminths do this better because they hide all their body)

Question 21

Schistosoma background

Answer 21

Flatworm trematode- romantic coupling while mating- causes Schistomosiasis
Common in tropics, subtropics, Subsaharan Africa, Latin America, Philippines, Indonesia, also in Surinam: often dutch patients come from Surinam
200 mill people infected
Only %10 symptomatic, rest asymptomatic
There’s many schistosoma species: 5 infective ones to humans: S.mansoni, S.japonicum, S. mekongi, S. intercalatum, S.hematobium
other species infect cattles,birds,reptiles etc

Question 22

Lifecycle Schistosoma

Answer 22

1) Eggs are released from human feces to water
2) Eggs come in contact with water, they swell and burst, release miracidium
3) Miracidium can swim away/do chemotaxis, so they go to snail
They infect different snails, depending on the snail population, different Schistosomes are observed. Not all snail is found everywhere, eg. Japonicum can only grow in Asia bcs of snail intermediate host type
4) Miracidium develops inside the snail, in 4 weeks, %50 of snails biomass is parasite wow.
5) releases cercaria everywhere, few mm, hardly visible, its tail does squeezing movements allowing it to move, it does chemotaxis to the skin of humans
6) it attaches to bare feet often in water, salivary glands of cercaria release proteases, which develops a hole inside the final host
Once cercariae enter, its head enters, tail falls off
7) Cercaria either migrates to the bladder or intestine depending on the type of Schistosoma and finds its mate and cuddles for sexual reproduction
8) Human lays produced eggs, cycle continues

How it travels to GI or urinary tract: Skin - vessel- lung capillaries- liver- bladder and intestine. So it goes from blood for sure. They develop as they move through I guess. They look like a cm long visible cables, they are very big for vessels actually. And those worms can survive up to 15-30 years, despite all immunity forever not recognized. It has two mouth suckers and moves w them.

Question 23

Schistosoma lifecycle

Answer 23

1) Eggs are secreted from humans, once they come in close contract with water, they swell and burst
2) Miracidium is released: they can swim and chemotaxis to find snails
3) Miracidium finds the snail and starts developing inside it, in 4 weeks it covers %50 body mass of the snail
4) Snail releases cercaria everywhere, few mm, hardly visible, it also moves around with its tail movements, so it does chemotaxis to skin
5) once it finds host w barefoot, it attaches and salivary glands release proteases that drills a hole in final host, head enters and tail falls off
6) Depending on schisto, it either goes to bladder or intestine, finds its mate, and cuddles for true love = sexual reproduction
7) then human lay more eggs and continues the cycle
Their movement to urinary or GI tract: Skin-blood vessels-capillaries of lungs- liver-then bladder or intestine = so it spends most of its time in blood.
You can see them with eyes, a cm of larvae moving like a cable inside vessels, its actually very big for vessels. As cercariae moves, it develops into larvae btw.
These worms survive up to 15-30 years without complete clearance

Question 24

Schistosoma pathogenesis

Answer 24

Problem: eggs gets stuck in tissues, as time passes, gets more stuck= cause tissue damage
%50 of the eggs can only be secreted
In blood vessels: eggs also attach to the platelets/endothelia causing coagulation, immune cells come there but can’t do much about it: they are too big to phagocytose, crosslinked eggs cant be degraded
Also in tissues: too much immune response drives granuloma formation
Since they also go to the liver: they cause liver fibrosis and failure, liver tissue dies a lot = so this swelling is observed, liver and spleen tries to save the problem by expanding = people end up having huge organs
Treatment: Praziquantel, but it cant reverse tissue damage

Adults do not cause many pathologies, can survive for years, inhibit the immune response like crazy

Question 25

How does Schistosome adults survive?

Answer 25

Check Helminth - Hermelijn Smit class!

Question 26

Host vessel/trombocytes worm/eggs interaction

Answer 26

The worm is giant inside the human vessel, if male and female would move together they cover the whole vessel (%80 covered)
Blood flow is disturbed, but no clotting occurs = they apparently cleave the coagulation factors around, they interfere with coagulation a lot, but bleeding happens

Platelets bind massively to especially eggs, which allows eggs to bind epithelia
also might allow them to penetrate to tissues
clotting is also driven because eggs and trombocytes accumulate together

Worms don’t bind thrombocytes, there’s some interaction but no binding, if they would bind adults, huge thrombosis and immune response would occur

So adults: don’t bind blood cells=no coagulation
Eggs. bind, cause coagulation

Question 27

The outer shell of Schistosoma mansoni: teguments

Answer 27

Crosssection: weird outer surface, shell-like, also progresses beneath w vesicles
The extra layer is called tegument (it has continuous layer of fused cells), its crucial for survival + also has double membrane
It protects from MAC + complement = Ab sticks on these but cannot make the membrane leaky, because of that shell around
Vesicles transform things outside to continuously refresh this layer, and its present in other blood helminths too

To study teguments: Isolate + LC+ mass spec/proteome analysis
LC -Mass/spec checks lipids: and a study showed that lipid composition of tegument is really different from rest of the worm and the blood cells (also SDS page shows tegument worm proteins are different)
It has lysophospholipids: phospholipids w only 1 FA instead of 2. They act like detergent, and they make it flexible
It has: -more than %50 cholesterol = makes it rigid
-has saturated phospholipids
-has high lysophosp. = less rigid
-has weird fatty acids/weird double bonds/combinations
Those weird lipids do:
Stimulate IL-10, IL-6, TNFalpha
Lysophosp.: stimulate TLR2, more IL-10, reduces immune response/Tregs
-they have short half life, cannot be used for other therapoetics
Proteomics: showed many tegument specific proteins/not found elsewhere on worm, also some of them are either unique to Schisto or other blood trematodes
= Can’t blast them to see their function w different sequences, function unknown

Question 28

How schisto specifically survives in blood vessel for years?

Answer 28

Teguments* lysis protection
Mimicry: larvae cover themselves with host factors/glycans also similar
Immunomodulation: th-2, IL-10, Tregs w lysophospholipids
Adults prevent platelet formation