Article: Modulation of innate immunity by Toxoplasma gondii virulence effectors Flashcards
Lifecycle toxoplasma gondii
Class: Intracellular protozoan
Indirect cycle
1) In cat: sexual reproduction occurs, oocystes are formed, inside theres sporozoites
2) Sporozoites are released to the soil, either forming unsporulated oocysts and going to environment to infect other hosts
3) In intermediate host: it either goes tachyzoites: acute infection
or bradyzoites: chronic infection
4) In humans it invades gut, and spreads everywhere
T.gondii structure
-Has PV around - vacuole, protects from intracellular attacks
-Uses actin motility
-Has rhoptry proteins : mediates movement with RONs (neck), and once it enters intracellular: covered with ROP proteins (bulb)
ROP5, ROP18, ROP2 on bulb
ROP16 goes to nucleus
Immune response for T.gondii
1) First response: recognised by TLRs on DCs (11) /macrophages (2-4) : leading to secretion of IL-12 (and TNFalpha) -TLR deficiency: more susceptible
2) Adaptive response: IL-12 activates NK/T cells (NFkB) , they release IFN-gamma
3) IFNgamma response: IFNgamma binds to receptors: Activates STAT1 - ROS-NO/IRG upregulation to rupture the cell
IFNγ alters host cell metabolism:
- tryptophan degradation in fibroblasts
- iron starvation in enterocytes
ROP overview
Rhoptry protein kinase 18: Active serine-threonine kinase (they phosphorylate certain downstream regulators on the pathways to activate them) that covers the surface of this PV membrane
If you express ROP18 in low expressing strains like type III here, it shows the increase in virulence directly, therefore it’s very responsible for virulence.
Rhoptry protein kinase 16: it goes to nucleus, therefore it alters the gene transcription.
Rhoptry protein pseudokinase 5: Again the knockout of its gene lead to severe reduction in virulence (as you remember pseudokinases don’t really phosphorylate, but they rather act as scaffolds to keep some regulators around)
ROP5 and 18 work together: and those two genes explain nearly %90 of the virulence differences in different strains.
Strain information
According to the mice studies:
Type I: highly virulent, uniformly lethal with the infection of a single organism
Type II: intermediate virulence (varies depending on mouse strain)
Type III: considered avirulent
Expression of virulence factors differs greatly between strains
But these are the most studied ones/ most common in America and Europe
The infections in humans are mainly seen due to the Type II strains, and this strain is abundant in life stock too.
Specifically, type II strain is used to study to understand the intracellular pathogens, remember they drive the pathogen into bradyzoites, and this strain both shows the acute and chronic response.
ROP16
To see the role of ROP16: they checked the host gene expression. And they mainly focused STAT3 and 6 here.
Although every strain phosphorylates STAT3 and 6 and activates it initially, only type I and type III strains continue this phosphorylation.
So this ROP16(⅓) found on the vacuole are used by the pathogen to phosphorylate and activate STAT, and this drives IL-4 expression rather than IL-12.
limits Th1 response → Less inflammation, reduced pathology, enhanced parasite survival
Type II strain ROP16 (II) has a single substitution in ROP16 that causes the loss of long term STAT3/6 activation
Also if you force to express ROP16(⅓) strain in Type II strains, you rescue this response and downregulate IL-12. And also if you knockout this, IL-12 increases and drives TNF-alpha. So what this means is, if you do not have this specific Type I-III ROP16, STAT phosp. does not occur, and downregulation of IL-12 does not happen.
But its hard to see its direct effect in the cell, Type I strain is very virulent and Type III is not, therefore it involves also other factors. And this is also reported in the article, it’s said that ROP16 has only a moderate effect in acute virulence. In fact if you delete the gene, it doesn’t really reduce the mortality in Type I strain, and rather it grows a bit faster.
The reason for this is slight increase in growth is because:
especially the STAT6 activation leads to production of arginase and,
it provides polyamines with the degradation of arginine (positive effect on growth)
you remove the essential aminoacid arginine from around (negative effect on growth)
BUT it grows a little bit faster overall since you see also an impairment in macrophages.
because this STAT3/6 activation leads to alternatively activated macrophages
once the arginine is reduced in these macrophages, nitric oxide (NO) cannot be produced, So by reducing their reactive oxygen species, you reduce their deadliness.
GRA15
If you infect cells with Type II strains, it shows the most IL-12 response.
GRA15 is shown to be responsible for this. Also if you express this GRA15(II) in Type I strain, that also induces IL-12. So you need specifically the Type II version.
We don’t know the exact mechanism of how GRA15 works, but here’s the general scheme we know:
Once macrophages get infected with GRA15 type II strain, they switch to the classically activated macrophage phenotype. So IL-12 induced pathway leads to more of a pro-inflammatory phenotype with more cytokines and chemokines with more T helpers, but the previous alternative pathway lead to more of a anti-inflammatory phenotype-M2 like.
So depending on which strain you are infected with, your macrophages tend to respond differently.
ROP18/ROP5
Other virulence factors that are critical for different strains are the serine threonine kinase ROP18 and pseudokinase ROP5.
These factors distrupt the host immunity related GTPases (IRGs), they are simply GTPases that turn GTP into GDP to produce energy for the pathways of immunity. Those GTPases were recruited by the IFN-gamma pathway for parasite response. The previous virulence factors ROP16 and GRA15 was more involved here in IL-12 production, now we will be talking about IFN-gamma response.
So in Type II and III strains, GTPase recruitment here leads to the death of the parasite, since immune system forms a vesicle around it and kills it. But Type I strain blocks this recruitment, and they survive. If you express this type I ROP18 in type III strain, you can also prevent their death
There are three different versions of GTPases, but for our discussion Irga6 and Irgb6 is important.
Apparently, ROP18 Type I phosphorylates the GTPases, and for example, on Irga6, GTPase and oligomerization is diminished.
Therefore you can’t place it on the vesicle.
So type I ROP18 increases the survival because the parasite doesn’t get destroyed. This is both seen in phagocytes as well as fibroblast like cells.
Also there’s this transcription factor here, activating transcription factor six-beta (ATF6beta).. Normally this TF is involved in degrading misfolded proteins. Here I put an ER because actually this protein is in ER .ROP18 phosphorylates it to be targeted for degradation. The reason why this factor is degraded its apparently because it has a role in antigen presentation, it just leads to secretion of more IFN-gamma.
If you knock ROP18 out in vitro, Type I parasite is highly attenuated, but not so attenuated if you knock this out in vivo.
But if you delete ROP5 gene, the virulence in Type I strain attenuates more than 1 million fold.
Usually pseudokinases don’t do much like we said, but here: what actually happens is that ROP5 changes the conformation of Type I ROP18 and obtains a different kinase that is more active, so it only binds to Irga6 but not the Irgb6.
Type I
What you need to remember: Type I strain of Toxoplasma gondii has:
high levels of ROP18
and this ROP18 cooperate with Type I ROP5. This ROP5 does 1) prevent the loading of GTPase to the vesicule as I showed, and the parasite does not get degraded 2) it also degrades that ER protein I showed you
ROP16 in this strain is also sufficient
GRA15 doesn’t activate NFkB, so no IL-12, no M1/no Thelper/more anti-inflammatory
So all this leads to death in mice. Although apparently mice produces a lot of Type I cytokines, but due to this impaired T helper response they cannot stop the parasites.
Type II
Type II strain of Toxoplasma gondii has:
High ROP18
But they have a non-virulent type of ROP5 that does not induce a conformational change in ROP18= so they cannot prevent the vesicule formation
This version has Type II GRA15, which drives IL-12 with NF-kB signaling = it gives proinflammatory response, and you can fight with infection
This supports the observation of tachyzoites, since mostly Type II strain infects the humans, this response can make it chronic.
Type III
Finally: the least virulent strain:
In this strain,they activate STAT3 and 6, so the pathway does not go to NFkB, so no IL-12, no overproduction of cytokines.
possibly, like I mentioned in very beginning of the introduction if you upregulate this ROP18 virulence should increase significantly. Again ROP5 you can alter it to be more virulent, those are the %90 of all the virulence.
