TEM analysis of knob appearance of the P. falciparum IE

The performed TEM analysis aimed the phenotypical differences of the IT4 culture isolate, cultivated at different conditions, focusing the overall knob appearance and distribution. The comparison with various field isolates completed the picture regarding the physiology of natural infections.

6.12.1.1 Field isolates

Here, distinct field isolates were analyzed, also including the cultivation with RPMI, complemented with human serum and AlbuMAX. As expected, all isolated exhibited a knobby phenotype, which is common in natural infections. Albeit, a recent study could show the appearance of knobless IE within field isolates, here, no knobless IE were detected in any sample. Moreover, the shown cultivation, either in the presence of human serum or AlbuMAX did not alter the number of detected knobs on IT4 IE, while significantly within 3D7 isolate.¹⁴² Here, the differential cultivation induced no significance change in knob appearance. However, as the field isolate BNI111216 was already processed 2 days after cultivation, it serves as baseline for physiological statements, regarding the number of detected knobs. Surprisingly, both, the BNI220616_HS and BNI220616_ALB isolate reached comparable levels, while being in culture for about 2 months. While the BNI060616_long isolate, cultivated for a similar time span, exhibited a fewer ratio of detected knobs per µm. While only being adapted for about 14 days, the other isolates show a clearly smaller number of detected knobs, while only the BNI010916 still reach the physiological baseline. The numbers of detected knobs on the IE surface for culture isolates were slightly higher, but in the same range and thus in line with prior findings.¹⁴²

6.12.1.2 Culture isolate IT4

Regarding the culture isolate IT4, cultivated at different temperatures and conditions or enriched over various endothelial receptors, the results differ from those of field isolates. While the IT4-St population exhibit a stable knobless phenotype, as evidenced by several samples and time points, there are some conditions, promoting the detection of knobby IE within the populations. By artificial selection for knobby IE via enrichment by Gelafundin, 100% of the population present a knobby surface, already after the first round of enrichment. The IT4 populations, after enrichment over human endothelial receptors were compared. As almost no receptor binding result in the presence of a knobby phenotype, it was somewhat surprising that IT3-CD37 and

DISCUSSION

key marker protein for knobby phenotypes, the transcription levels should be elevated in knob positive, while almost undetectable in knob negative populations. By comparing former NGS analysis of the IT4 isolate, enriched over WT, CD36, ICAM-1, CD9, CD151, CD62-E and CD62-P and similarly processed as the actual experimental setup, the expression levels for KAHRP were concerned.¹⁷⁵ Thus, the levels, detected in the IT4-WT and IT4-CD151 population range in with the levels for IT4-MDR1, IT4-CD55 and IT4-CD81. The levels for IT4-CD36, IT4-CD9 and IT4-CD62-P were in the same range as IT4-TNFR1 and IT4-TNFR2. The outstanding expression level of IT4-CD37 is positively related to the knob appearance, visible in the TEM analysis, while 83% of the population exhibit a knobby phenotype. As also 12% of the IT4-CD81 population display knobs, while KAHRP being in the same range as other enriched, but knobless population, it is logical to assume that not KAHRP alone mediate knob formation on IE surfaces. Therefore, also, IT4-WT and IT4-CD151 IE should have at least some detectable knobby IE within their population, while they don’t.

Comparing the results for the temperature dependent experimental setups, it was striking that only the IT4-40°C IE, enriched over HBEC-5i at 40°C, exhibited a knobby phenotype, even in 100% of the population. Otherwise, IT4-37°C, enriched over HBEC-5i at 37°C did not. Moreover, IT4 cultivated at elevated temperatures, experienced reoccurring febrile heat shocks or a treatment with TNFa exhibited no knobby phenotypic IE at all.

Therefore, my question rose, if the process of sequestration, compared with febrile temperatures is a decisive model to generate knobby phenotypes within IE populations. To test the newly set thesis, 2 enriched IT population should be compared regarding their phenotypic expression after experiencing sequestration and febrile temperatures. To rule out the involvement of HBEC-5i cells, triggering the expression, sequestration was induced towards mock-transfected CHO-GFP cells, and CHO-CD55 cells, expressing the respective human endothelial receptor. Therefore, the equally enriched IT4-GFP population, as well as the IT4-CD55 population was co-incubated or rather preabsorbed by either 37°C or 40°C, followed by the actual panning at 40°C.

Within this setup, also the time frame of febrile temperatures was adjusted, to see if an abrupt temperature change (37°C to 40°C) or a continuous temperature (40°C to 40°C) is more favorable. By immunofluorescence analysis utilizing a specific αKAHRP antibody, knobby IE could be detected for both, the IT4-GFP and IT4-CD55 population, within the temperature changing approach. Consequently, these results could also be validated in the TEM analysis. (data not shown) Otherwise, the continuous elevation of the temperature prior and at the panning, was proved not to be successful.

Taking to account that long-term cultivated isolates are capable of chromosomal deletions and genomic alterations, experimental setups could be established, using adapted field isolates that have lost or reduced their knobby phenotype over the time.

Also, analyzing the genomic background of the individual population could be easily tested with particular qPCR panels, focusing knob related genes. The possibility that only knobby IE were positively selected via the enrichment procedure is given, albeit not likely. The initial knobless IT4-CD55 population does express KAHRP in the same range as the knobby IT4-CD81 population, until enrichment at febrile temperature, where knobby IE can be detected.

Moreover, it would be interesting if the initial knobby phenotype of IT4-CD37 and IT4-CD81 could be altered into a knobless one by enrichment over other human endothelial receptors e.g. TNFR1, which enriched populations do not exhibit knob structures, such as IT4-TNFR1. A former study showed that knobless IT4 populations, expressing A_var60 can switch to a knobby phenotype, when enriched for E_var04 (low knob density) or B_var32B (high knob density) expression, and vice versa. Here, the theory rose that the differentially expression of a particular var gene alter the knob related phenotypic appearance.¹³⁹ However, those findings are not applicable to the actual experiments, as all enriched population highly express the B_var32B gene, while consequently being knobless. The only exemptions are IT4-CD37, while no particular differential upregulated var genes was found; and IT4-CD81, differentially expressing A_var09, B_var11 and B_var12; also found in other populations. However, it could be of interest to validate if the expression profile is still the same before and after an enrichment at febrile temperatures of 40°C. There might be the possibility, that some var genes are more favored to be expressed at elevated temperatures, coupled with endothelial sequestration.

> As these experiments were only caries out one, a repetition is clearly needed. Also, further controls could be the implementation of a knobby isolate, such as 3D7, to evaluate if also there, the overall amount or the density of knobs increase. As the gene expression level for KAHRP is nearly not detectable for the enriched IT4-GFP population, for IT4-CD55, it was in the same range as the initially knobby IT4-CD81 population.

Similar levels are detected for IT4-MDR1, while no TEM analysis or IFA is available. This would be a potential condition investigating the knob appearance as well. Also, other enriched population could be further processed at 40°C panning assays, evaluating the possibility of generating knobby phenotypes in distinct population, or if the appearance is restricted to the sequestration of few selected receptor proteins.

Therefore, it would be interesting to utilize other cell lines, such as HEK cells or primary cell lines to draw a more physiological picture and to determine if the effect can be restored with unspecific binding as well.

> To clarify the involvement of particular genes in the formation of knob structures, it would be advisable to compare whole NGS analysis of the general knobless isolate IT4 and the partly knobby isolate 3D7. As the data for both isolates are available and include the starting population, as well as the enriched populations, a comparison of all knob related and maybe so far undetermined genes could be drawn. This would give a high impact on understanding and evaluating the available expression levels, at relatively low further costs and effort.