An unusual trafficking domain in MSRP6 defines a complex needed for Maurer’s clefts anchoring and maintenance in P. falciparum infected red blood cells

Reviewer #1 (Public review):

Summary:

In this paper, Blancke Soares and Stäcker et al serendipitously identify a domain of the Plasmodium falciparum protein MSRP6 that mediates both export from the parasite into the infected red blood cell and association with the Maurer's cleft organelles found in the infected cell. The authors use this domain to identify a putative complex of proteins at the Maurer's cleft via proximity biotinylation. Six members of the complex are confirmed to interact with MSRP6 by co-immunoprecipitation.

The functions of select proteins of this complex are further investigated with regard to the formation of Maurer's clefts. Disruption of PeMP2, PIESP2, and Pf332 resulted in morphological changes to the Maurer's clefts and prevented the anchoring of the Maurer's clefts to the infected red blood cell plasma membrane that normally occurs in the trophozoite stage. Curiously, disruption of MSRP6, the central member of the complex, did not affect Maurer's cleft anchoring. Mechanistically, how this complex affects Maurer's cleft structure and anchoring remains unclear.

Finally, the authors show that the loss of Maurer's cleft anchoring observed upon disruption of PIESP2 or Pf332 does not affect cytoadherence of infected red blood cells via PfEMP1, arguing against a prior assumption that cleft tethering is required for the presentation of parasite-exported proteins on the infected red blood cell surface.

Strengths:

Maurer's clefts are enigmatic organelles found in red blood cells infected by Plasmodium falciparum that are presumed to play a role in trafficking exported parasite proteins to the surface of the red blood cells, though little is known about their biogenesis and function. The authors here convincingly identify a protein complex present at the Maurer's clefts using multiple orthogonal tools, and carry out assays that indicate this protein complex has a role in shaping and anchoring the Maurer's clefts at their final location at the red blood cell membrane. The data indicating that Maurer's cleft anchoring is dispensable for trafficking of P. falciparum exported proteins to the infected red blood cell membrane has implications for understanding the function of this organelle.

Weaknesses:

In many instances, the data lack appropriate controls that would be desirable for the highest level of rigor. Many, if not most, fluorescence microscopy assays lack untagged/parental controls (prepared in parallel and captured with the same settings) that are necessary to determine the validity of the data - that the observed signal is specific to the protein of interest and not due to autofluorescence or bleed-through from other channels. In other cases, wild-type controls are missing where data from disruption mutants are presented. Additionally, while some phenotypes are quantified, others are only qualitatively described where a more thorough quantitative investigation would be valuable. Finally, where phenotypes have been quantified, in many instances it is not clear that the analyses have included biological replicates as would be expected.

Reviewer #2 (Public review):

Summary:

Soares et al characterize several P. falciparum exported proteins that localize to Maurer's Clefts (MCs), membrane structures formed in the host erythrocyte cytosol. MCs are thought to act as sorting stations that mediate the trafficking of effector proteins to the erythrocyte membrane, such as the surface adhesin and major virulence factor PfEMP1. While initially mobile within the host cytosol, MCs become anchored at the erythrocyte periphery around the time PfEMP1 appears on the RBC surface. While MC immobilization is thought to be important for the delivery of PfEMP1 onto the erythrocyte surface, this hypothesis has remained untested due to the lack of mutants that prevent anchoring. The study begins by determining the sequence features able to mediate the export of PF3D7_0830300 and MSRP6, both PEXEL-Negative Exported Proteins (PNEPs) with signal peptides. The authors show that in both proteins, a region downstream of the signal peptide is sufficient to mediate export, indicating the mature N-terminus is also important for the translocation of this type of PNEP, similar to other classes of exported proteins. Surprisingly, an additional C-terminal region of MSRP6 is also sufficient to mediate export when placed downstream of the signal peptide in the absence of other MSRP6 features. This region also mediates recruitment to MCs and was used as BioID bait to identify proximal MC proteins, several of which form a complex with MSRP6. Strikingly, disruption of certain MSRP6 interacting proteins (PeMP2, PIESP2, and Pf332) abolishes MC anchoring and in some cases also results in major changes in MC morphology. Surprisingly, neither PfEMP1 surface display nor cytoadhesion of infected RBCs is impacted in these mutants. This study features an impressive array of genetically modified parasites and will be of broad interest in providing the first functional analysis of MC anchoring, challenging the prevailing model for PfEMP1 trafficking within the infected RBC.

Strengths:

(1) The first section of the paper presents an in-depth dissection of the features that enable the export of signal peptide-containing PNEPs, confirming the mature N-terminus is sufficient for export across all known types of exported proteins. While it remains unknown how these features enable export, the results reinforce the universal importance of the mature N-terminus, whether generated by signal peptidase or Plasmepsin 5.

(2) The discovery that a C-terminal region of MSRP6 (MAD) is also sufficient for export is novel. The authors suggest this may be the result of piggybacking on another exported protein, although the discussion acknowledges there are challenges with this model since unfolding by PTEX would be expected to disrupt these interactions. An alternative might be considered: the related protein MSRP7 is also exported but consists essentially of a signal peptide and MSP7-like domain without the large N-terminal region found in MSRP6. Presumably, the mature N-terminus of MSRP7 mediates export. If MSRP6 is derived from an exported predecessor composed only of the MSP7-like domain (like MSRP7), the MAD domain might retain the ancestral export information near the beginning of the MSP7-like domain. If this were the case, then the MAD domain (3cd region) should only be sufficient to mediate export when positioned immediately after the signal peptide as in the experiment in Fig 3C (SP-3cd-GFP). It would be interesting to determine if an SP-GFP-3cd construct is exported.

(3) Disruption of PeMP2, PIESP2 or Pf332 is found to prevent MC anchoring. This is the most exciting part of the study as it provides the first set of mutants that interfere with anchoring, enabling the surprising observation that MC immobilization is not important for PfEMP1 surface display or cytoadhesion. The MC movement assay is a nice way to visualize anchoring and would be strengthened by a quantitative measure of colocalization between the time-lapse images (ie, Pearson correlation coefficient) to enable a statistical test. The use of SLI to specifically activate a var gene of choice is an exciting new approach that will be of great use to the PfEMP1 field together with the semi-automated binding assay that helps to increase throughput and reduce bias.

Weaknesses:

(1) At least two of the MSRP6 complex members were found to depend on other complex members for MC trafficking: PeMP3 depends on MSRP6 and Pf332 depends on PIESP2 (previously shown by Zhang et al 2018 and confirmed in the present study). While the authors disrupted all seven MSRP6 complex members, the impact on the trafficking of the other complex members was not systematically investigated. It would be particularly interesting to know which (if any) complex members are required for MC recruitment of PeMP2 since this protein is also needed for MC anchoring.

(2) Some images of exported puncta are interpreted as localization to the MCs without a co-marker. Since other compartments have been identified in the RBC cytosol in addition to MCs (ie, J dots), an MC co-marker would help to verify these actually correspond to MCs. For example, in Figure 5B, GEXP18 gives an exported punctate appearance but lack of co-localization with SBP1 in Fig S2B shows that this does not correspond to MCs.

(3) The authors show MAHRP2 localization is not impacted in their PIESP2 and Pf332 mutants and this is interpreted to indicate the tether structures are not disrupted. However, this conclusion requires actual analysis of the tether structures by electron microscopy since MAHRP2 association to MCs may not require tether integrity and could persist even if the tethers are altered or disrupted. Otherwise, this statement should be adjusted. Additionally, since T2A skipping efficiency can vary between constructs, it would be a good idea to perform a western blot to ensure that the SBP1-GFP and MAHRP2-mScarlet signals in Figure 8D,F reflect separated proteins.

(4) The trypsin assays to monitor PfEMP1 surface display would benefit from a more detailed explanation of how the results were interpreted. For instance, though perhaps less intense than in the PIESP2, Pf332, and MSRP6 mutants, a Var01-protected fragment is also seen in the SBP1 mutant. Additionally, a protected fragment is indicated for most of the SBP1N controls (asterisk). As per the author's experimental design (lines 956-957), does this indicate that the RBC membrane was partially compromised during the experiment? In line 505, the trypsin assay data in the mutants is interpreted relative to the parent IT4var01-HA line but no data is shown for the parent.

Reviewer #3 (Public review):

Summary:

Malaria is caused by Plasmodium falciparum parasites that infect, grow, and reproduce inside red blood cells. The parasites extensively modify the blood cells they infect, by exporting hundreds of proteins into the red blood cell compartment. One of the most important modifications made by the parasite is to display adhesive proteins on the blood cell surface which attach the infected cells to walls of small blood vessels. This can lead to organ damage resulting in serious disease complications and there is great interest in blocking the adhesive process to reduce disease. This study investigates the function of an atypical, exported protein that along with other proteins maintains the integrity of membranous sacs formed by the parasite in the blood cell compartment. These sacs are widely believed to help organise the display of the adhesive proteins on the infected blood cell surface. This study challenges this dogma by showing that disruption of the sacs does not prevent the display of the adhesive proteins suggesting alternative pathways are likely involved in adhesive protein display.

Strengths:

The conclusions are supported by a beautiful series of live parasite images.

Weaknesses:

No major weaknesses were identified by this reviewer.