Essential function reflected in the phylodynamics of a multigene family – the pir genes of malaria parasites
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This valuable study provides the first broad cross-species evolutionary analysis of the pir multigene family in malaria parasites, showing that the family evolved through rapid duplication and loss while retaining a small number of conserved orthologs with essential functions. The authors identify pirC1 as a key determinant of parasite growth across multiple Plasmodium species. However, the work remains incomplete because the mechanistic role of PIRCl and its precise subcellular localization are not directly resolved.
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Abstract
The genomes of malaria parasites ( Plasmodium spp .) encode many gene families, which are intimately associated with host interactions and disease in these important pathogens. The largest malaria gene family is the Plasmodium interspersed repeat ( pir ) genes, present in rodent, primate and most human malaria parasites, which are suggested to have originated from one highly conserved gene, which we call pirC1 . The precise function(s) of pir is unknown but to determine their potentially multifarious roles we must understand the evolutionary dynamics of pir repertoire to discriminate among the many genes. Here we estimate the global phylogeny for pir genes in 14 Plasmodium species and one Hepatocystis species. We reveal that pirC1 is not the common ancestor but is one of several orthologous genes conserved in multiple species amidst the rapid turnover of species-specific paralogs. We show that the PIRC1 protein is nonetheless essential for blood stage growth of P. berghei, P. chabaudi and P. knowlesi , as parasites lacking the pirC1 gene could not be generated or had severely reduced growth rates. As this effect was observed both in vivo and in vitro, the role of pirC1 is not related to host immune interaction. Rather, P. berghei and P. knowlesi PIRC1 are secreted from the parasite, pointing to a role in parasite interaction with the host cell or nutrient uptake by blood stages. The phylodynamics of pir genes indicate that old orthologs, like pirC1 , and younger within-species paralogs could have fundamentally different roles, and emphasize the need to distinguish between them in future. This study is the first to provide evidence for the existence of an essential pir gene and provides a robust rationale for further experimental approaches to pir gene functions.
SIGNFICANCE
The genomes of malaria parasites ( Plasmodium ) contain many different gene families, of which the pir family is the largest, with more than 1000 members in some species. The PIR proteins are likely important for parasite fitness but their precise functions remain unknown – roles in adherence of infected red blood cells to blood ves sels, virulence and immune evasion of have been suggested. How, and why, this highly diverse gene family evolved is a significant question both for understanding malaria physiology and pathogenesis. Here we present a comprehensive pir phylogeny, identifying the origins of gene diversity during Plasmodium evolution and a select group of highly conserved genes. We show that one conserved pir gene ( pirC1 ) encodes a protein that is essential for optimal growth of multiple malaria parasites during the asexual blood stage, both in the host and in vitro. This indicates that pirC1 function relates to interaction with the host cell or nutrient acquisition, and not to immune evasion or sequestration, (although this might still be the function of other pir genes). This study provides a robust rationale for the hitherto baffling diversity of pir genes, and shows why it is important to distinguish old orthologs from young paralogs in future studies on pir gene function.
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eLife Assessment
This valuable study provides the first broad cross-species evolutionary analysis of the pir multigene family in malaria parasites, showing that the family evolved through rapid duplication and loss while retaining a small number of conserved orthologs with essential functions. The authors identify pirC1 as a key determinant of parasite growth across multiple Plasmodium species. However, the work remains incomplete because the mechanistic role of PIRCl and its precise subcellular localization are not directly resolved.
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Reviewer #1 (Public review):
Summary:
The manuscript entitled "Essential function reflected in the phylodynamics of a multigene family - the pir genes of malaria parasites" by Jackson and colleagues investigates the global phylogeny of pir genes across 14 Plasmodium species and one Hepatocystis species. The authors also focus on the functional characterization of the conserved ortholog pirC1 and claim that pirC1 is not the founder of the family and that it plays an essential role in blood-stage growth.
Strengths:
Overall, the manuscript is well written and interesting, as it combines comparative genomics and evolutionary analysis with functional experiments. The phylogenetic analysis is rigorous and represents a major strength of the manuscript.
Weaknesses:
The general conclusions regarding the potential function of this gene family are …
Reviewer #1 (Public review):
Summary:
The manuscript entitled "Essential function reflected in the phylodynamics of a multigene family - the pir genes of malaria parasites" by Jackson and colleagues investigates the global phylogeny of pir genes across 14 Plasmodium species and one Hepatocystis species. The authors also focus on the functional characterization of the conserved ortholog pirC1 and claim that pirC1 is not the founder of the family and that it plays an essential role in blood-stage growth.
Strengths:
Overall, the manuscript is well written and interesting, as it combines comparative genomics and evolutionary analysis with functional experiments. The phylogenetic analysis is rigorous and represents a major strength of the manuscript.
Weaknesses:
The general conclusions regarding the potential function of this gene family are not fully supported by the data presented. The manuscript moves too quickly from growth phenotype and localization studies to a specific mechanistic model. The discussion argues that PIRC1 may be involved in nutrient acquisition, host sensing, or metabolic support, but the data provided do not directly support these functions, and the manuscript in its present form remains speculative. Although the manuscript includes some experimental results, it lacks direct mechanistic validation of the specific functions of the pir genes, including pirC1. In its current form, the study does not yet establish a definitive role for pirC1 in metabolic processes.
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Reviewer #2 (Public review):
Summary:
This is an extensive study using phylogenetic comparison across multiple plasmodium species to gain new insights in relation to their evolutionary pathways and the potential function of pir. In addition to establishing a framework to identify related orthologues across species as well as expanding paralogues families within a species, the work also focuses on understanding loss and gain of different PIRs and how this indicates a relative lack of functional constraints and essentiality for most members of the gene family.
The authors provide evidence that at least pirC has a conserved function and plays an important role in parasite growth in multiple species.
While this study represents a significant effort and does provide interesting new insights that would help our understanding of this complex …
Reviewer #2 (Public review):
Summary:
This is an extensive study using phylogenetic comparison across multiple plasmodium species to gain new insights in relation to their evolutionary pathways and the potential function of pir. In addition to establishing a framework to identify related orthologues across species as well as expanding paralogues families within a species, the work also focuses on understanding loss and gain of different PIRs and how this indicates a relative lack of functional constraints and essentiality for most members of the gene family.
The authors provide evidence that at least pirC has a conserved function and plays an important role in parasite growth in multiple species.
While this study represents a significant effort and does provide interesting new insights that would help our understanding of this complex gene family in the future, it has a number of limitations.
Strengths:
Extensive and thorough phylogenetic analysis that is supported by some biological validation. Provides an indication that the PIR gene family has limited biological constraints and evolved independently across different species, leading to rapid expansion and deletion of orthologous groups. Identified pirC as a functional and important member of the family that is conserved across the species.
Weaknesses:
The phylogenetic tree is based on a truncated sequence that focuses on the more conserved parts of the pir sequence. This could potentially lead to missing the key functional drivers of evolution. The biological validation of the role of pirC has some inconsistencies that need to be addressed.
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Reviewer #3 (Public review):
This paper aims to classify, from an evolutionary perspective, the multigene family PIR found in malaria parasites infecting rodents and Old World monkeys, and to link this classification to functional diversification. The authors also hypothesize that PIR members conserved across species play important roles in parasite survival, and seek to clarify their functions.
To achieve these aims, the authors comprehensively analyze the evolution of PIR genes using genomic and transcriptomic information from many malaria parasite species. They focus on PIRC1, a member conserved across species, and attempt to clarify its function in rodent and simian malaria parasites by examining the phenotypes of parasites in which the corresponding genetic locus has been disrupted. They also attempt to determine its localization …
Reviewer #3 (Public review):
This paper aims to classify, from an evolutionary perspective, the multigene family PIR found in malaria parasites infecting rodents and Old World monkeys, and to link this classification to functional diversification. The authors also hypothesize that PIR members conserved across species play important roles in parasite survival, and seek to clarify their functions.
To achieve these aims, the authors comprehensively analyze the evolution of PIR genes using genomic and transcriptomic information from many malaria parasite species. They focus on PIRC1, a member conserved across species, and attempt to clarify its function in rodent and simian malaria parasites by examining the phenotypes of parasites in which the corresponding genetic locus has been disrupted. They also attempt to determine its localization using PIRC1 tagged with an epitope sequence. However, although the locus-disrupted parasites appear to show an approximately 50% reduction in growth rate, this effect seems to be overestimated. Another weakness is that the cause of the reduced growth rate has not been clarified. The localization analysis also remains insufficiently conclusive.
Therefore, I consider that the first half of the paper, consisting of the bioinformatics analyses, achieves the objective of comprehensively summarizing PIR and may become a reference paper for discussing the evolution and function of the PIR gene family. On the other hand, regarding the function of PIRC1, no clear conclusion can be drawn from the results presented, and several additional experiments are necessary.
My major comments are as follows.
(1) The claim that the failure of eight disruption attempts indicates that pirC1 is essential is too strong.
Lines 319-321: The authors argue that a total of eight failed attempts to disrupt the pirC1 locus using two different construct designs suggest that pirC1 is essential in P. berghei. However, the failure of these attempts could also reflect technical issues with the construct design itself, such as the length of the homologous regions used for recombination, which are approximately 650 bp. Therefore, it is an overstatement to conclude that "pirC1 is essential for P. berghei blood-stage growth." Given that parasites with disruption of the corresponding locus could be obtained in both P. chabaudi and P. knowlesi, a more appropriate statement would be that "pirC1 is important for P. berghei blood-stage growth."
(2) The data on the mCherry-expressing P. berghei line shown in Supplementary Figure 11 are insufficient.
(a) Panel C: Southern blot analysis
To conclusively identify the lower band in panel C as chromosome 1, additional probes specific to genes located on chromosomes 1 and 2 would be required. In addition, a parental parasite control should also be included. The Southern blot image of the parental parasite should show only a single band at the higher position, with no band at the lower position. Probes specific to chromosomes 1 and 2 would help demonstrate that the lower band corresponds to chromosome 1, rather than chromosome 2.To this end, the authors could describe the result as follows:
"In the parental parasite, only a single band corresponding to chromosome 7 was detected, indicating that the smaller chromosome was genetically modified. The size of the lower band detected with the dhfr probe was identical to that of the band detected with the control chromosome 1 probe, but distinct from that detected with the chromosome 2 probe, indicating that chromosome 1 was modified."That said, this chromosome-level Southern blot analysis is not sufficient to demonstrate that the target PBANKA_0100500 locus was specifically modified. The authors should provide more direct evidence showing that the PBANKA_0100500 locus, rather than another genomic locus, was modified. For example, Southern blot analysis after restriction enzyme digestion would provide more definitive evidence. Diagnostic PCR may also provide more specific evidence.
(b) Panel D: Flow cytometry analysis
To allow a more accurate interpretation of the percentage of mCherry-positive cells, flow cytometry data for the parental parasite line should also be presented.
(3) There are unclear points in the PCR results shown in Supplementary Figure 12.
Supplementary Figure 12: In panel B, a PCR product should also be amplified from dPCHAS_0101200 using the P1-P3 primer pair. Why is this band absent? The authors should provide the uncropped electrophoresis image so that the larger band can be seen. In addition, if labels 1 and 2 indicate independent clones, this should be stated in the figure legend.
(4) The growth rates of P. chabaudi and P. knowlesi parasites with disruption of the PIRC1 gene locus should be quantitatively analyzed.
The growth rates of P. chabaudi and P. knowlesi are described only qualitatively, but they should be evaluated quantitatively. In Figure 4A, the parasitemia of wild-type P. chabaudi increases from approximately 6.1% on day 6 to approximately 15.6% on day 8, corresponding to a 3.8-fold increase. However, because parasite growth may already be affected by immune-mediated suppression at this stage, this value should be regarded as a minimum estimate. In contrast, the mutant increases from approximately 3.2% on day 8 to approximately 6.8% on day 10, corresponding to a 2.1-fold increase. Based on these values, the daily growth rate of the mutant appears to be reduced to at least approximately 56% of that of the wild type. Similarly, from the growth curve of P. knowlesi in Fig. 5A, the DMSO-treated group appears to increase approximately two-fold per day, whereas the rapamycin-treated group increases only approximately one-fold per day. Thus, P. knowlesi also appears to show an approximately 50% reduction in growth rate. Taken together, both P. chabaudi and P. knowlesi appear to reproducibly show an approximately 50% reduction in growth capacity. A reduction of this magnitude is difficult to describe as a "severe growth defect"; a more appropriate wording would be simply that the parasites "showed a growth defect." In addition, the terms "a severe growth defect" and "essential" appear to be overstated throughout the manuscript, and the wording should be toned down. Finally, I recommend presenting Figure 4A and Figure 5A on a logarithmic scale so that the trend in growth rates can be more intuitively appreciated from the graphs.
(5) The evidence that disruption of the PIRC1 gene locus in P. knowlesi does not affect erythrocyte invasion is weak.
The authors describe that "the developmental cycle of the parasites lacking PIRCl is slightly longer than that of parasites that produce PIRCl (line 383-384)," and appear to support this interpretation with data showing that "mutant parasites are significantly smaller than wild-type parasites (line 414)" and that "the DNA content in ML10-arrested parasites lacking PIRCl is lower than that of DMSO-treated parasites (line 417-418)" at 24 hours after invasion. However, a slightly longer developmental cycle alone does not seem sufficient to explain a 50% growth reduction.
I think the erythrocyte invasion capacity has not been quantitatively evaluated, and therefore, the evidence supporting the conclusion that the phenotype of P. knowlesi parasites with disruption of the PIRC1 gene locus is unrelated to erythrocyte invasion is weak. The authors should assess invasion efficiency using purified merozoites. For P. chabaudi, it should also be possible to apply an in vitro or in vivo erythrocyte invasion assay similar to that used for other rodent malaria parasites, and this should be evaluated as well.
(6) The authors should examine whether disruption of the PIRC1 gene locus results in a phenotype characterized by a reduced number of merozoites.
Alternatively, the reduced DNA content in ML10-arrested parasites lacking PIRC1 (lines 416-417) could suggest that the number of merozoites formed per schizont may be reduced. To clarify this point, the authors should assess whether the number of merozoites per schizont is altered in P. knowlesi (and P. chabaudi parasites lacking PIRC1).
(7) The authors propose the possibility that PIRC1 expressed in merozoites is released after invasion; however, the evidence that PIRC1 localizes to intracellular organelles is weak.
Line 333: "a peripheral pattern around the parasite" is indicative of parasite plasma membrane, PV, or PVM. ", indicative of a parasitophorous vacuole (PV) or parasitophorous vacuole membrane (PVM) location" should be amended to ", indicative of parasite plasma membrane, a parasitophorous vacuole (PV) or parasitophorous vacuole membrane (PVM) location". In the Figure S14 image, red signals are uniformly detected from the merozoites formed in the schizont stage parasite (not really microorganelle patterns), but not from the PVM surrounding the schizont, suggesting parasite plasma membrane localization, not PVM. I agree that the signal is detected from the compartments extending into the iRBC cytosol, which may be difficult to explain if it is located on the parasite plasma membrane, but how frequently were such images seen?
Figure 4D. In the images of liver-stage schizonts, AMA1 does not appear to localize to the micronemes in mature merozoites, suggesting this image is an immature schizont. Although PIRC1 appears to be expressed in liver-stage schizonts, it is difficult to clearly determine whether it localizes to intracellular organelles or to the parasite plasma membrane.
To clarify the above points, the authors should examine whether PIRC1 is detected in intracellular organelles or around the merozoites by analyzing its localization in purified merozoites.
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