IT4-TNFR2

For IT4-TNFR2 a similar profile as IT4-MDR1 was seen. While for A_var09 a significant upregulation was calculated, while also, here, only C_var05 met the cutoff.

C_var05

DBLα0.5 CIDRα2.3 DBLδ1 CIDRβ1 See description for IT4-MDR1 above.

A_var09

DBLα1.6 CIDRγ3 DBLγ15 DBLε1 DBLδ1 CIDRβ1

A_var09 is one of the about 20% of var genes, not harboring a CD36 binding head structure. However, regarding the first domain, DBLα1.6 is not associated with SM.²⁴⁸ As for CIDRγ3 no clinical importance was described, the tandem structure of DBLγ15 and DBLε1 can be found in group upsA type var genes, generally associated with SM and CM.¹⁴⁵ Additionally, A_var09 is also differentially upregulated in IT4-CD9 enriched population.¹⁷⁵ During flow cytometry binding assays and bead-associated binding assays towards endothelial cell types of various tissues, A_var09 bound at very low or negligible levels and displayed low and patchy binding to different.²⁴⁹ The B_var09 is also differentially upregulated in IT4-CD81. Also, here, the TNFR2 involvement sequestration was already described and proven by specific antibodies inhibition of IE of the IT4 and the MM field isolate.¹⁴⁶ Nevertheless, a repetition of this experiment would reveal also the specificity obtained within this study.

Other genes

For IT4-TNFR2 quiet a lot of genes exhibit a differential upregulated expression level, surpassing the chosen cutoff criteria. Regarding the overall picture, genes involved in knob formation processes and protein transport towards the IE membrane surface are

ETRAMP, the GYP binding protein and 2 FIKK kinases, all 3 HSP coding genes, as well as MAHRP1, PfEMP2/MESA, almost all PHIST coding genes of all subgroups, 3 RIFIN, RESA-like (involved in KAHRP self-assembling), SURFIN8.1 and SURFIN8.2, as well as all members of the VSP and HYP family. This intense conglomerate of related proteins gives a strong indication of sequestration assignment of IT4-TNFR2 binding IE. The enhanced activation of transport, trafficking and membrane alteration seems to be additionally evidenced by the upregulated gene expression levels of up to 22 different conserved membrane proteins with unknown function. TNFR2 was investigated in the same course as TNFR1 and determined as putative interaction partner of P. falciparum IE, while also being associated with CM in patients.146,198,200,201

6.10.5 IT4-CD37

For IT4-CD37, B_var11 and E_var04, or var2csa show a slight tendency of regulation, albeit below the cutoff criteria.

Other genes

Albeit no unique upregulated var gene could be detected within the IT4-CD37 enriched population, the results of the whole transcriptome give a striking evidence of knob formation processes, mediated by IE binding towards the CD37 protein. The highly elevated levels of the acyl-CoA synthetase, 2 PHIST_a, the RESA-like PHIST and 2 PHIST_b, SBP1 and the surface adhesion associated SURFIN1.2 give a hint of an excited protein trafficking towards the IE membrane and their modification.²⁵⁰ Strikingly, the knob determined DnaJ (~115fold), KAHRP (163fold) and the knob associated HSP40 (~200fold), PfEMP3 (~65fold) and the knob associated NA-type RIFIN are of remarkably interest, due to their extended upregulation, compared to the IT4-ST population. This overall view strongly suggests and underpins the presence of knobs on the IE surface.

Also, here, only a few genes, coding for conserved membrane proteins with unknown function were detected above the cutoff. Regarding the effect associated within the rodent malaria P. yoelii and as CD37 can build complexes with CD81, an interaction of those proteins or even a synergistic effect regarding sequestration is quite possible.¹⁸³

6.10.6 IT4-CD55

Regarding IT4-CD55, a significant upregulated expression level was calculated for B_var11.

B_var11

DBLα0.3 CIDRα2.4 DBLβ10 DBLδ1 CIDRβ1

The CD36 binding head structure is followed by a, so far, barely determined DBLβ10 domain and the DC4 clustered tandem domain, already described above for IT4-MDR1. The B_var11 is also differentially upregulated in IT4-CD81. The specificity of the CD55 involvement of sequestering IE was determined by utilizing specific antibodies against CD55, prior a static binding assay, albeit only one trial was

DISCUSSION

performed. Nevertheless, a repetition of this experiment, additionally with various antibody concentrations, would affirm the obtained results.

Other genes

Applying the cutoff criteria for the IT4-CD55 enriched population, only few genes exhibited an expression level over the chosen cutoff, such as a DnaJ, a A-type RIFIN, the surface adhesion associated SURFIN1.2, VSP53, a HYP12 and both HYP17 and just a few conserved membrane proteins with unknown function hit the level. ²⁵⁰ Nevertheless, within the IT4-CD55 enriched population, also KAHRP was differentially upregulated, giving a hint towards the presence of knob structures on the IE surface, albeit no other related genes surpassed the cutoff. Unexpectedly, also invasion and merozoite associated genes were differentially affected within this population, such as EBA-181 and members of the RBP family. This phenomenon could rather be traced back to a certain amount of merozoites within the processed sample, than to a physiological reason, regarding the actual CD55 binding. As CD55 is described to be a critical receptor for Plasmodium merozoite committed attachment and invasion to host erythrocytes, those results could be misleading. While no correlation of the observed genes was drawn to IE sequestration, nor was the protein expression abundant in the mid-trophozoites stages. Nevertheless, it is possible that proteins, expressed in merozoites can be injected or introduced into the host erythrocyte. If not destroyed or degraded, they can be stored or traverse to the IE membrane, exercising a second function, like a task related to later staged parasitized IE. This phenomenon could be already shown for STEVOR variants and other proteins, like RIFIN and the rhoptry-derived antigen. This may contribute significantly to the long-term survival of the parasite by an efficient management of the natural resources.96,124,262–265 Even not relevant within this study, this thought could be considered regarding further experiments.

6.10.7 IT4-CD81

For IT4-CD81, a significant upregulation was calculated for A_var09, while B_var11 and B_var12 reached the cutoff.

A_var09

DBLα1.6 CIDRγ3 DBLγ15 DBLε1 DBLδ1 CIDRβ1 See description for IT4-TNFR2 above.

B_var11

DBLα0.3 CIDRα2.4 DBLβ10 DBLδ1 CIDRβ1 See description for IT4-CD55 above.

B_var12

DBLα0.18 CIDRα6 DBLβ4 DBLδ1 CIDRβ1

The CD36 binding head structure is followed by a, so far, barely determined DBLβ4 domain and the DC4 clustered tandem domain, already described above for

IT4-MDR1. Interestingly, the B_var12 is additionally differentially upregulated in a significant manner in IT4-MDR1, while not surpassing the cutoff level.

Other genes

For IT4-CD81, some merozoite and invasion assigned genes surpassed the cutoff criteria, indicating a certain amount of merozoites within the processed samples. Those genes comprise the EBA, MSP and RBP members. Furthermore, the gamete release protein also exceeded the cutoff, maybe giving a hint on elevated levels of gametocytes or sexually committed parasites, albeit no further evidence was found.

Interestingly, and in somehow similar to the profile of IT4-CD37, most of the knob related genes show a differentially upregulated gene expression level, such as the acyl-CoA synthetase, the PfEMP1 trafficking associated AG 332, the lysine-rich PHIST_b and RESA.

Moreover, also, ETRAMP10.2, a FIKK kinase, PfEMP2/MESA, the RESA-like (involved in KAHRP self-assembling), 2 PHIST and 3 RIFIN, 2 VSP and – solely within this population – CLAG 3.1 exhibited elevated levels. Surprisingly, completely all 6 SURFIN and all members of the HYP family were differentially upregulated, while they do not share the premise of mutually exclusive expression.^124,125 However, only the SURFIN13.1 also showed a differentially upregulation in IT4-CD9 and IT4-CD62-P of within a prior study.¹⁷⁵ CD81 is also known to be used by P. falciparum, P. vivax, P. yoelii parasites to enter human or murine hepatocytes with formation of a parasitophorous vacuole.^182,184 However, no correlation of sequestrating IE was drawn so far, leaving the hypothesis of similar activated genes in two different stages of the parasitic life cycle, where same proteins can outlast the whole 48 h cycle and undertake a task related to cytoadhesion or membrane modifications at later stages. Matching, up to 22 genes for conserved membrane proteins with unknown function are differentially upregulated within the IT4-CD81 population. Also, an interesting theoretical hypothesis was triggered by a recent study, regarding the likelihood of activated var gens.¹³² Here, it was described that no var gene is in a determined active state, all have the same probability of being activated. This status is valid at the point in the life cycle, when parasites first exit the infected liver. When merozoites firstly circulating within the blood flow, random var gene activation by each individual ensure an antigenically diverse population. Examination of experimental human infections have detected activation of all var genes immediately after parasite leave the liver, consistent with this model. Moreover, here, it was indicated that in cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but upsA group var genes were transcribed at the lowest levels. However, cultures established from second or third generation blood stage parasites expressing specific var genes, mainly belonging to group upsA and upsB, therefore being more effectively in vivo compared to parasites expressing other var genes.²⁶⁶ As CD81 represent a prominent invasion receptor for P. falciparum parasites into human hepatocytes, the binding towards this protein could trigger a change in var gene expression, also in blood stage parasites. A rearrangement in var gene expression hierarchy could be triggered, maybe by changing the 'molecular memory' archive by e.g. the alteration of specific histone modifications via particular activation of methyltransferases. The modification due to sirtuin-like PfSIR2 proteins is not likely, as the respective genes do not exhibit any

DISCUSSION

receptor, it is assigned to merozoite invasion and not hepatocyte attachment. The even though slight enhanced expression levels of upsA group var genes is only observed within the IT4-CD81 population, may give a hint towards this theory.