Cell surface localisation of GPI-anchored receptors in Trypanosoma brucei

Reviewer #1 (Public review):

Summary:

An interesting manuscript from the Carrington lab is presented investigating the behavior of single vs double GPI-anchored nutrient receptors in bloodstream form (BSF) T. brucei. These include the transferrin receptor (TfR), the HpHb receptor (HpHbR), and the factor H receptor (FHR). The central question is why these critical proteins are not targeted by host-acquired immunity. It has generally been thought that they are sequestered in the flagellar pocket (FP), where they are subject to rapid endocytosis - any Ab:receptor complexes would be rapidly removed from the cell surface. This manuscript challenges that assumption by showing that these receptors can be found all over the outer cell body and flagella surfaces, if one looks in an appropriate manner (rapid direct fixation in culture media).

The main part of the manuscript focuses on TfR, typically a GPI1 heterodimer of very similar E6 (GPI anchored) and E7 (truncated, no GPI) subunits. These are expressed coordinately from 15 telomeric expression sites (BES), of which only one can be transcribed at a time. The authors identify a native E6:E7 pair in BES7 in which E7 is not truncated and therefore forms a GPI2 heterodimer. By in situ genetic manipulation, they generate two different sets of GPI1:GPI2 TfR combinations expressed from two different BESs (BES1 and BES7). Comparative analyses of these receptors form the bulk of the data.

The main findings are:

(1) Both GPI1 and GPI2 TfR can be found on the cell body/flagellar surface. (2) Both are functional for Tf binding and uptake. (3) GPI2 TfR is expressed at ~1.5x relative to GPI1 TfR. (4) Ultimate TfR expression level (protein) is dependent on the BES from which it is expressed.

Most of these results are quite reasonably explained in light of the hydrodynamic flow model of the Engstler lab and the GPI valence model of the Bangs lab. Additional experiments, again by rapid fixation, with HpHbR and FHR, show that these GPI1 receptors can also be seen on the cell surface, in contrast to published localizations.

It is quite interesting that the authors have identified a native GPI2 TfR. However, essentially all of the data with GPI2 TfR are confirmatory for the prior, more detailed studies of Tiengwe et al. (2017). That said, the suggestion that GPI2 was the ancestral state makes good evolutionary sense, and begs the question of why trypanosomes prefer GPI1 TfR in 14 of 15 ESs (i.e., what is the selection pressure?).

Strengths and weaknesses:

(1) BES7 TfR subunit genes (BES7_Tb427v10): There are actually three (in order 5'-3'): E7gpi, E6.1 and E6.2. E6.1 and E6.2 have a single nucleotide difference. This raises the issue of coordinate expression. If overall levels of E6 (2 genes) are not down-regulated to match E7 (1 gene), this will result in a 2x excess of E6 subunits. The most likely fate of these is the formation of non-functional GPI2 homodimers on the cell surface, as shown in Tiengwe et al. (2017), which will contribute to the elevated TfR expression seen in BES7.

(2) Surface binding studies: This is the most puzzling aspect of the entire manuscript. That surface GPI2 TfR should be functional for Tf binding and uptake is not surprising, as this has already been shown by Tiengwe et al. (2017), but the methodology for this assay raises important questions. First, labeled Tf is added at 500 nM to live cells in complete media containing 2.5 uM unlabeled Tf - a 5x excess. It is difficult to see how significant binding of labeled TfR could occur in as little as 15 seconds under these conditions. Second, Tiengwe et al. (2017) found that trypanosomes taken directly from culture could not bind labeled Tf in direct surface labeling experiments. To achieve binding, it was necessary to first culture cells in serum-free media for a sufficient time to allow new unligated TfR to be synthesized and transported to the surface. This result suggests that essentially all surface TfR is normally ligated and unavailable to the added probe. Third, the authors have themselves argued previously, based on binding affinities, that all surface-exposed TfR is likely ligated in a natural setting (DOI: 10.1002/bies.202400053). Could the observed binding actually be non-specific due to the high levels of fixative used?

(3) Variable TfR expression in different BESs: It appears that native TfR is expressed at higher levels from BES7 compared to BES1, and even more so when compared to BES3. This raises the possibility that the anti-TfR used in these experiments has differential reactivity with the three sets of TfRs. The authors discount this possibility due to the overall high sequence similarities of E6s and E7s from the various ESs. However, their own analyses show that the BES1, BES3, and BES7 TfRs are relatively distal to each other in the phylogenetic trees, and this Reviewer strongly suspects that the apparent difference in expression is due to differential reactivity with the anti-TfR used in this work. In the grand scheme, this is a minor issue that does not impact the other major conclusions concerning TfR localization and function, nor the behavior of HpHbR and FHR. However, the authors make very strong conclusions about the role of BESs in TfR expression levels, even claiming that it is the 'dominant determinant' (line 189).

(4) Surface immuno-localization of receptors: These experiments are compelling and useful to the field. To explain the difference with essentially all prior studies, the authors suggest that typical fixation procedures allow for clearance of receptor:ligand complexes by hydrodynamic flow due to extended manipulation prior to fixation (washing steps). Despite the fact that these protocols typically involve ice-cold physiological buffers that minimize membrane mobility, this is a reasonable possibility. Have the authors challenged their hypothesis by testing more typical protocols themselves? Other contributing factors that could play a role are the use of deconvolution, which tends to minimize weak signals, and also the fact that investigators tend to discount weak surface signals as background relative to stronger internal signals.

(5) Shedding: A central aspect of the GPI valence model (Schwartz et al., 2005, Tiengwe et al., 2017) is that GPI1 reporters that reach the cell body surface are shed into the media because a single dimyristoylglycerol-containing GPI anchor does not stably associate with biological membranes. As the authors point out, this is a major factor contributing to higher steady-state levels of cell-associated GPI2 TfR relative to GPI1 TfR. Those studies also found that the size/complexity of the attached protein correlated inversely with shedding, suggesting exit from the flagellar pocket as a restricting factor in cell body surface localization. The amount of newly synthesized TfR shed into the media was ~5%, indicating that very little actually exits the FP to the outer surface. In this regard, is it possible to know the overall ratio of cell surface:FP:endosomal localized receptors? Could these data not be 'harvested' from the 3D structural illumination imaging?

Reviewer #2 (Public review):

The work has significant implications for understanding immune evasion and nutrient uptake mechanisms in trypanosomes.

While the experimental rigor is commendable, revisions are needed to clarify methodological limitations and to broaden the discussion of functional consequences.

The authors argue that prior studies missed surface-localized TfR due to harsh washing/fixation (e.g., methanol). While this is plausible, additional evidence would strengthen the claim.

It remains unclear how centrifugation steps of various lengths (as in previous publications) can equally and quantitatively redistribute TfR into the flagellar pocket. If this were the case, it should be straightforward for the authors to test this experimentally.

If TfR is distributed over the cell surface, live-cell imaging with fluorescent transferrin should be performed as a control. Modern detection limits now reach the single-molecule level, and transient immobilization of live trypanosomes has been established, which would exclude hydrodynamic surface clearance as a confounding factor.

In most images, TfR is not evenly distributed on the surface but rather appears punctate. Could this reflect localization to membrane domains? Immuno-EM with high-pressure frozen parasites could resolve this question and is relatively straightforward.

The authors might consider discussing whether differences in parasite life cycle stages (procyclic versus bloodstream forms) or culture conditions (e.g., cell density) affect localization. The developmentally regulated retention of GPI-anchored procyclin in the flagellar pocket might be worth mentioning.