On 2019-10-31 06:38:40, user Prem Prakash wrote:
Heartiest congratulations to Stachwell and co-workers for this seminal and groundbreaking study that was, as stated above, subsequently published as: "Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements" (Nature Communications, 10: 3806; 2019; https://www.nature.com/arti....
This response pertains to the Stachwell et al observation on the human Cyclophilin B (CypB) receptor in relation to parasite invasion and their report that the knockout of cyclophilin B in their reticulocyte cell-line derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line BEL-A (that caused 20% less expression of GPC) resulted in "no significant difference" in the invasive susceptibility of reticulocytes.
As the primary author of the 2017 Nature Communications paper that reported the role of Human Cyclophilin in the parasite invasion process (https://www.nature.com/arti..., it is believed the following observations would add to the discussion and analysis surrounding the seminal Stachwell study:
It is to be noted that Stachwell et al state (https://static-content.spri... that they have not tested either the anti-malarial drug and Cyclophilin inhibitor Cyclosporin A (CsA) or CDP3, the peptide inhibitor of CypB, on their WT and CypB KO BEL-A lines, because, as they emphasise, neither CsA nor CDP3 are specific to cyclophilin B over other cyclophilins. CsA binds to multiple cyclophilins, and CDP3 binds to cyclophilin A and perhaps other cyclophilins. Therefore, as Stachwell et al observe: "Our CypB KO line contains multiple known binding partners for these drugs, which would convolute the results of this experiment."
Furthermore, Stachwell et al state (https://static-content.spri... that they found "CsA to be poorly soluble, making such experiments practically challenging. Finally, BEL-A derived reticulocyte production and subsequent parasite invasion assays represent a significant time and cost investment, which, given the specificity and solubility concerns, we decided to devote to other experiments to improve the manuscript."
We believe that, in addition to being an important advance in Malaria research, the findings of Stachwell et al are entirely in keeping with the nature of Cyclophilin inhibition that was reported by us in 2017 (https://www.nature.com/arti..., for the following reasons:
A. Cyclophilins, the Cyclosporin A (CsA) binding proteins and their role in malaria pathogenesis.<br />
Cyclophilins were first identified as binder of Cyclosporin A (CsA) and hence named cyclophilins [1-4]. In humans, there are 16 Cyclophilins reported till today. Of these, Cyclophilin A (CypA) was the first cyclophilin discovered in mammalian host and is the most prevalent followed by the Cyclophilin B (CypB) [2, 5].
CypA and CypB share ~80% sequence similarity overall, and 70% identity in their core region [6]. CypB is found on the surface while CypA has been found to be secreted outside of the cell in response to inflammatory responses and oxidative stress [7-10].
When CsA binds to Cyclophilins, they form an intracellular ternary complex involving Calcineurin [11]. CsA potently blocks the enzymatic activity of cyclophilins [11]. Furthermore, extracellular CypB has been reported to mediate the incorporation of CsA in T-lymphocytes [12]. Also, because the structure of CypB and CypA is almost identical, the CsA-binding pocket in CypB has the same structure as in CypA, with CsA exhibiting similar conformation and network of interactions when it is bound to these proteins [11, 13-15]. Yet another study shows that cyclophilins were identified as erythrocyte cyclosporine-binding proteins [16]. Finally, in addition to our observation that CypB is found on the surface of the RBC, a recent study confirms the presence of CypA and CypB in the RBC ghost fraction through LC-MS/MS [17].
The antimalarial activity of CsA was identified as early as in 1981 when it was shown that CsA inhibits the parasite invasion in human cells in vitro and in vivo in a mice model [18, 19]. In another report, low dose treatment with CsA showed inhibitory effect on the mice cerebral malaria pathogenesis [20]. These studies indicate that the mode of action of CsA as antimalarial is through binding to the Cyclophilins.
Basigin has been identified as the extracellular receptor for CypA and CypB [23]. The interaction between Cyclophilins and Basigin has been implicated in many medical conditions including inflammatory responses, cardiovascular disease, and rheumatoid arthritis [1]. The interaction of cyclophilins with Basigin on the cell surface has been proven by utilising monoclonal antibodies against Basigin. Treatment of CHO cells with anti-Basigin antibodies inhibits the Cyclophilin-induced chemotaxis response [21, 23].
Summary: CsA binds to Cyclophilin B and this binding is responsible for the potent anti-malarial activity of the drug. Cyclophilin binds to Basigin.
B. Our Study I:<br />
1. Our 2017 finding (https://www.nature.com/arti..., that cyclophilin B is a host receptor for PfRhopH3 starts with the discovery of human cyclophilin B as an interacting partner of PfRhopH3-C through screening a human lung c-DNA library using the bacterial two hybrid system (B2H). Further, in our study, this interaction has been confirmed through a range of in vitro techniques like ELISA, SPR, Far-Western Blot, Co-IP and an in vivo B2H β-galactosidase assay. (Figure 1b, c, Figure 3a, b, c, d)
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Subsequently, to confirm that PfRhopH3 is a ligand for CypB, we performed binding and co-localisation studies with recombinant CypB on the surface of merozoite. Reciprocally, binding and co-localization of PfRhopH3-C was also studied on the RBC surface. (Figure 1d, e; Figure 2a, b; Figure 3e; Supplementary fig 2d, e)
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Basigin has earlier been reported as a receptor/interacting partner, both for PfRH5 as well as CypB. Therefore, to confirm this we performed IFA based expression and co-localization of CypB and Basigin on the RBC surface and found that these two proteins are present together on the RBC surface. Reciprocally, Basigin and CypB bind on the merozoite and are co-localized. (Figure 4a, b, c)
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To establish the significance of CypB and PfRhopH3 during invasion, we then looked for a peptide inhibitor from a de novo peptide library and fished out CDP3 as a binder of CypB. CPD3 was tested for the disruption of interaction of PfRhopH3-C and CypB using Bacterial Three Hybrid System (B3H) and ELISA-based competitive inhibition assay. In both experiments CDP3 was found to potently disrupt the interaction. (Figure 5; Supplementary fig. 7)
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Notably, as CypA and CypB are found to be sharing 70% sequence identity in their core region, we obtained the recombinant CypA protein. Utilising the ELISA based interaction experiments, the interaction of CypA with RhopH3-C as well as CDP3 was tested and it was found that CypA can also interact with these two molecules very strongly. (Supplementary fig. 12)
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Following on, we tested the effect of CDP3 on invasion through two different assays. In the first, CDP3 was added to 2% haematocrit with 1% tightly synchronised schizont stage of 3d7 and Dd2 culture. It was observed that parasitaemia decreased in response to an increasing dose of CDP3. In the second, the RBCs were first treated with different doses of CDP3 and these RBCs were then used for the invasion assay. In this experiment, we again observed a dose dependent reduction in parasitaemia. (Figure 6a, b)
Summary: CypB is expressed on the RBC surface. Both CypB and CypA – because of their sequence identity – interact not only with RhopH3C but also with the CDP3 peptide. CDP3 reduces parasitaemia in a dose-dependent manner.
C. Our study II: Cyclosporin A and malaria invasion<br />
1. As stated above, it is well-established that Cyclophilin is the target protein and binder for Cyclosporin A (CsA). To confirm the role of host cyclophilins in invasion, we used CsA, first, to treat the RBC for 1 h (https://www.nature.com/arti.... Unbound CsA was washed out extensively and the treated RBCs were then used for invasion assay. In the second experiment, CsA was introduced directly to culture. In both assays, a significant reduction in the parasitaemia was observed, suggesting that host Cyclophilins (CypB and CypA) are directly involved in the parasite invasion. Notably, when CsA was directly added in the culture, reduction in parasitaemia was more for the same concentration as compared to the washed RBC. This observation suggest that CsA potentially blocks the host cyclophilins (CypA, CypB) as well as parasite Cyclophilins. (Figure 6c, d, e). This is a crucial point, in that the drug CsA, that binds to CypB, blocks invasion when it is introduced either i) to the RBC first, and ii) to a culture.<br />
2. In our in vivo experiments, when CsA is administered to the Balb/C mice followed by challenging of the latter with the parasite, we found growth to be completely absent in treated mice as compared to the untreated mice (Supplementary Fig. 11a). All the untreated mice died by day 20 whereas the treated mice survived beyond day 30 (Supplementary Fig. 11b). This observation corroborates the original finding that CsA acts an antimalarial drug targeting the Cyclophilins [18-22].
Summary: Based on our experimental findings and previous reports, host cyclophilins play a role during parasite invasion. Small molecules like CsA and CDP3 can potentially block these receptors on the cell surface and prevent the parasite entry in isolation or together with their complex.
D. Our explanation in view of the Satchwell et al report: <br />
1. There are as many as 16 host cyclophilins out of which, just to give one example, CypB shares an overwhelming similarity (80% overall) and idenditity (70% in the core region) with CypA [24]. It is entirely conceivable, therefore, that in the absence of CypB, other cyclophilins – most notably CypA, whose copy number, it should be emphasised, is greater than CypB in RBC ghost [17] – have taken its place for carrying out its receptor-mediated role. And while CRISPR/Cas9 has removed completely from the system only CypB, all other cyclophilins are still in place.
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Additionally, while the CRISPR KO has removed only CypB, inhibitors of Cyclophilins like CsA and our peptide CDP3 (that we have shown binds also to CypA) bind and incapacitate all Cyclophilins, thereby preventing the swapping of blocked (KOed) Cyclophilin with the unblocked one.
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This is brought out by the observation that CsA, when introduced to the RBC, and the latter then washed, blocks the invasion potently; meaning that Cyclophilins (one, maybe more, maybe all) are important for the invasion process because the drug that binds to them for its potent activity goes on to kill the parasite because of this very binding.
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As Satchwell et al have themselves stated (https://static-content.spri... neither CsA nor CDP3 are specific to cyclophilin B over other cyclophilins. CsA binds to multiple cyclophilins, and CDP3 binds to cyclophilin A and other cyclophilins. Therefore, their CypB KO line contains multiple known binding partners for these drugs. It stands to reason that if both CsA and CDP3 have multiple cyclophilin targets, a knock-out of just one of sixteen available cyclophilins would not have the desired effect on invasive susceptibility.
E. References:<br />
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8. Price, E.R., et al., Cyclophilin B trafficking through the secretory pathway is altered by binding of cyclosporin A. Proc Natl Acad Sci U S A, 1994. 91(9): p. 3931-5.<br />
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20. Grau, G.E., D. Gretener, and P.H. Lambert, Prevention of murine cerebral malaria by low-dose cyclosporin A. Immunology, 1987. 61(4): p. 521-5.<br />
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