Biology subjects

Swearingen, K. E.

Publications and source records attributed to Swearingen, K. E..

10 recordsLinked to original sources

bioRxiv2025

Glycosylation of Plasmodium falciparum TRAP is essential for infection of mosquito salivary glands.

The human malaria parasite Plasmodium falciparum (Pf) expresses ten different thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. PfTRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr256 and C-mannosylated at Trp250. We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two PfTRAP glyco-null mutant parasites, PfTRAP_T256A and PfTRAP_W250F, and assessed the fitness of these mutant parasites across the life cycle compared to the wild-type NF54 line as well as a PfTRAP knockout line. The PfTRAP glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. PfTRAP abundance in these mutants was significantly decreased despite normal transcript levels. Biophysical assays with recombinant proteins confirmed that glycosylation of the PfTRAP TSR stabilizes the domain and is likely required for its folding and secretion. These findings demonstrate that glycosylation of PfTRAPs TSR is critical for its proper expression and function, and underscore the importance of TSR glycosylation in the mosquito stage of the life cycle. IMPORTANCEMalaria is a mosquito-borne disease caused by Plasmodium parasites, of which P. falciparum is the deadliest. Plasmodium has ten proteins bearing thrombospondin type 1 repeats (TSRs), protein folds that aid cell-cell recognition and binding. Each of Plasmodiums ten TSR-bearing proteins is important for invading tissues in the mosquito vector and human host. TSRs are decorated with sugar molecules, a modification termed glycosylation. To better understand the importance of TSR glycosylation in Plasmodium, we investigated the P. falciparum protein TRAP, which is only expressed in mosquito-stage parasite forms called sporozoites. When PfTRAP was mutated to prevent glycosylation, abundance of the protein significantly decreased and parasites were unable to colonize the mosquito salivary glands. Furthermore, these mutant sporozoites were unable to move or to invade human liver cells. Our study reveals how TSR glycosylation can support the function of proteins that are required for parasite virulence.

cell biology↗

bioRxiv2024

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

biochemistry↗

bioRxiv2024

Global Release of Translational Repression Across Plasmodiums Host-to-Vector Transmission Event

Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5 or 3 end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.

microbiology↗

bioRxiv2023

Borrelia PeptideAtlas: A proteome resource of common Borrelia burgdorferi isolates for Lyme research

Lyme disease, caused by an infection with the spirochete Borrelia burgdorferi, is the most common vector-borne disease in North America. B. burgdorferi strains harbor extensive genomic and proteomic variability and further comparison is key to understanding the spirochetes infectivity and biological impacts of identified sequence variants. To achieve this goal, both transcript and mass spectrometry (MS)-based proteomics was applied to assemble peptide datasets of laboratory strains B31, MM1, B31-ML23, infective isolates B31-5A4, B31-A3, and 297, and other public datasets, to provide a publicly available Borrelia PeptideAtlas (http://www.peptideatlas.org/builds/borrelia/). Included is information on total proteome, secretome, and membrane proteome of these B. burgdorferi strains. Proteomic data collected from 35 different experiment datasets, with a total of 855 mass spectrometry runs, identified 76,936 distinct peptides at a 0.1% peptide false-discovery-rate, which map to 1,221 canonical proteins (924 core canonical and 297 noncore canonical) and covers 86% of the total base B31 proteome. The diverse proteomic information from multiple isolates with credible data presented by the Borrelia PeptideAtlas can be useful to pinpoint potential protein targets which are common to infective isolates and may be key in the infection process.

biochemistry↗

bioRxiv2022

Plasmodium GPI-Anchored Micronemal Antigen is essential for parasite transmission through the mosquito host

The complex life cycle of Plasmodium parasites, the eukaryotic pathogens that cause malaria, features three distinct invasive forms tailored specifically to the equally distinct host environment they must navigate and invade for progression of the life cycle. One conserved feature of all these invasive forms is the presence of micronemes, apically oriented secretory organelles involved in egress, motility, adhesion and invasion. Micronemes are tailored to their specific host environment and feature stage specific contents. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent infecting species Plasmodium berghei. While GAMA is dispensable during asexual blood stages, GAMA knock out parasites are severely defective for invasion of the mosquito midgut, resulting in reduced numbers of oocysts. Once formed, oocysts develop normally, however sporozoites are unable to egress and these sporozoites exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knock out parasites with full length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating a conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of the known ookinete-to-sporozoite stage-specific genes: CTRP, CAP380 and TRAP, further confirmed the involvement of GAMA in midgut infection, motility and infection of the mammalian host and revealed a lethal consequence to overexpression of GAMA during oocyst development. Combined, the research suggest that GAMA plays independent roles in sporozoite motility, egress and invasion, possibly implicating GAMA as a regulator of microneme function. AUTHOR SUMMARYMalaria remains a major source of morbidity and mortality across the globe. Completion of a complex life cycle between vertebrates and mosquitoes is required for the maintenance of parasite populations and the persistence of malaria disease and death. Three invasive forms across the complex lifecycle of the parasite must successfully egress and invade specific cell types within the vertebrate and mosquito hosts to maintain parasite populations and consequently disease and suffering. A conserved feature of all invasive forms are the micronemes, apically oriented secretory organelles which contain proteins required for motility, egress and invasion. Few proteins are expressed in the micronemes of all three invasive forms. One such protein is GPI-anchored micronemal antigen (GAMA). Here we reveal that GAMA is required for the invasion of the mosquito midgut, egress of sporozoites from oocysts and invasion of the vertebrate host. Our finding indicate that while GAMA is essential for sporozoite motility, the defects in oocyst egress and hepatocyte invasion occur independently of the motility defect, implicating the requirement of GAMA in all three processes.

microbiology↗

bioRxiv2021

Plasmodium falciparum Calcium Dependent Protein Kinase 4 is critical for male gametogenesis and transmission to the mosquito vector

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium dependent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4- parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4- parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild type and Plasmodium falciparum cdpk4- late gametocyte stages, to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events such as DNA replication, mRNA translation and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation, and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCETransmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

microbiology↗

bioRxiv2020

Platelet Derived Growth Factor Receptor β (PDGFRβ) is a Host Receptor for the human malaria parasite adhesin TRAP

Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor {beta} (hPDGFR{beta}) as one such protein receptor. Deletion mutants showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFR{beta}-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax, but not the rodent-infective parasite Plasmodium yoelii. We observed expression of hPDGFR{beta} mainly in cells associated with the vasculature suggesting that TRAP:hPDGFR{beta} interaction may play a role in the recognition of blood vessels by invading sporozoites.

microbiology↗

bioRxiv2019

Generating high-quality libraries for DIA-MS with empirically-corrected peptide predictions

Data-independent acquisition approaches typically rely on sample-specific spectrum libraries requiring offline fractionation and tens to hundreds of injections. We demonstrate a new library generation workflow that leverages fragmentation and retention time prediction to build libraries containing every peptide in a proteome, and then refines those libraries with empirical data. Our method specifically enables rapid library generation for non-model organisms, which we demonstrate using the malaria parasite Plasmodium falciparum, and non-canonical databases, which we show by detecting missense variants in HeLa.

systems biology↗

bioRxiv2019

Extensive Transcriptional and Translational Regulation Occur During the Maturation of Malaria Parasite Sporozoites

Plasmodium sporozoites are transmitted from an infected mosquito to mammals in which they infect the liver. The infectivity profile of sporozoites changes as they egress from oocysts on the mosquito midgut into the hemocoel, and then invade the salivary glands, where they maintain a poised and infectious state until transmission occurs. Upon transmission, the sporozoite must then navigate the host skin, vasculature, and liver. All of these feats require distinct repertoires of proteins and capabilities that are coordinated in an appropriate temporal manner. Here, we report the comprehensive and dynamic transcriptomes and proteomes of both oocyst sporozoite and salivary gland sporozoite stages in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. These data robustly define mRNAs and proteins that are Upregulated in Oocyst Sporozoites (UOS) or Upregulated in Infectious Sporozoites (UIS), which include critical gene products for sporozoite functions, as well as many of unknown importance that are similarly regulated. Moreover, we found that Plasmodium uses two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate specific genes necessary to successfully navigate the mosquito vector and mammalian host environments. Finally, gene-specific validation experiments of selected, translationally repressed transcripts in P. yoelii confirmed the interpretations of the global transcriptomic and proteomic datasets. Together, these data indicate that two waves of translational repression are implemented and relieved at different times in sporozoite maturation to promote its successful life cycle progression.

microbiology↗

bioRxiv2019

Proteomic Analysis of Plasmodium Merosomes: The Link Between Liver and Blood Stages in Malaria

The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for anti-malarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique dataset will allow us to explore key questions about the biology of merosomes and hepatic merozoites.\n\nHighlightsO_LIFirst proteome of the merosome stage of malaria parasites\nC_LIO_LIQuantitative detection of 1188 parasite proteins across 3 biological replicates\nC_LIO_LIComparison to blood stage proteomes identifies shared and unique proteins\nC_LIO_LIDiscovery of cleaved PEXEL motifs highlights liver stage protein export\nC_LI\n\nIn BriefThe merosome stage that links malaria liver and blood stage infection is poorly understood. Here we provide the first proteome of this life cycle stage using the Plasmodium berghei rodent malaria model.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=27 SRC=\"FIGDIR/small/580266_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (11K):\norg.highwire.dtl.DTLVardef@5bd8b1org.highwire.dtl.DTLVardef@1ef4fb9org.highwire.dtl.DTLVardef@5b3833org.highwire.dtl.DTLVardef@c69c73_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗