Biology subjects

King, C. L.

Publications and source records attributed to King, C. L..

8 recordsLinked to original sources

bioRxiv2026

The Unexpected Visibility of the SARS-CoV-2 Nucleocapsid Protein Reveals a Hidden Route of Surface Trafficking.

The SARS-CoV-2 Nucleocapsid (N) protein, long regarded as an internal structural component of the virion, unexpectedly localizes to the plasma membrane of infected cells. Here, we show that N is actively trafficked to the cell surface via a ceramide-dependent unconventional secretory pathway. Live-stained imaging and kinetic analyses revealed that N surface association begins early in infection, before Spike (S) expression and viral release, and persists after enzymatic removal of heparan sulfate. Pharmacological disruption of phosphoinositide or phosphatidylserine interactions had minimal effect, whereas inhibition of neutral sphingomyelinase with GW4869 markedly reduced surface N, identifying a ceramide-regulated route as essential for its export. This mechanism distinguishes N from canonical transmembrane viral proteins and explains how N-specific antibodies mediate potent Fc-effector responses across SARS-CoV-2 variants. Our findings redefine the spatiotemporal dynamics of coronavirus structural proteins and reveal an unanticipated axis of immune visibility within the infected cell. IMPORTANCEInternal viral proteins are generally thought to remain confined to intracellular compartments, yet several viruses display such proteins at the surface of infected cells through mechanisms that remain incompletely defined. In this study, we characterize a host-regulated trafficking route that contributes to the delivery of the SARS-CoV-2 nucleocapsid (N) protein, a non-membrane viral protein, to the plasma membrane. Our findings indicate that N protein surface expression occurs independently of virion assembly, membrane integration, or extracellular rebinding, and instead involves host vesicular processes outside the classical secretory pathway. By elucidating how a leaderless coronavirus protein can access the cell surface, this work addresses a key gap in coronavirus cell biology and provides a framework for understanding how internal viral proteins may exploit host trafficking pathways. More broadly, these results highlight unconventional host trafficking pathways as determinants of viral protein localization and provide a framework for understanding how viruses exploit cellular export mechanisms beyond the classical secretory system.

cell biology↗

bioRxiv2024

IgM plays a prominent role in naturally acquired immunity against Plasmodium falciparum gametocytes

The development of transmission-blocking vaccines against Plasmodium falciparum malaria could facilitate malaria elimination. However, limitations in the knowledge of the human immune responses against P. falciparum transmission stages, known as gametocytes, represent a critical roadblock to vaccine development. We evaluated human antibodies acquired through natural malaria exposure to whole gametocytes and recombinant antigens expressed by transmission stages, including the major transmission-blocking vaccine candidates Pfs230 and Pfs48/45 and other transmission stages, Pf38, Pf12 and Pf41. Among individuals residing in Kenya and Papua New Guinea, we found substantial antibody responses to whole gametocytes and to all recombinant transmission stage antigens with high levels of IgG, IgG subclasses and IgM. Complement fixation by antibodies to gametocytes is key for effective transmission-blocking activity. We found that purified IgM was substantially more potent than IgG at mediating complement fixation and activation. Higher antibody levels were generally observed in individuals positive for P. falciparum infection, including gametocyte positive individuals, and these antibodies generally increased with age. Our findings reveal that IgM is a prominent feature of antibody responses to gametocytes and that antibodies target multiple antigens. The further demonstration that IgM has high functional activity against gametocytes suggests IgM plays an important role in immunity to transmission stages. Our data provide new insights to inform the development of potent transmission-blocking vaccines.

immunology↗

bioRxiv2024

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. We isolated 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv-exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. HumAb 826827 blocked the invasion of human erythrocytes using Pv clinical isolates and inhibited sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 - 3.7 {micro}g/mL). It also significantly reduced liver infection of chimeric FRG-humHep mice in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate-transcendent, blocks both pre-erythrocytic and blood stage infection, and could be a new therapy for Pv.

immunology↗

bioRxiv2023

Naturally acquired and Vaccine-induced Human Monoclonal Antibodies to Plasmodium vivax Duffy Binding Protein Inhibit Invasion of Plasmodium knowlesi (PvDBPOR) Transgenic Parasites

The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to Plasmodium vivax (Pv) invasion of reticulocytes. Pv expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and their protein-protein interaction is central to vivax blood stage malaria. Here we compared the functional activity of humAbs derived from naturally exposed and vaccinated individuals for the first time using easily cultured P. knowlesi (Pk) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. Using this model, we evaluated the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 g/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 g/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10-100 g/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 g/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. This PkPvDBPOR model system enables efficient assessment of NA and VI humAbs individually and in combination. IMPORTANCEGiven the importance of Duffy blood group antigen and P. vivax Duffy binding protein (PvDBP) interaction leading to blood stage vivax malaria, development of vaccines or therapeutic human monoclonal antibodies (humAbs) targeting PvDBP are key strategies for treating and controlling Pv. The P. knowlesi-based PkPvDBPOR transgenic model system enables efficient assessment of NA and VI humAbs individually and in combination. As such, this model could prioritize specific humAb combinations ahead of clinical trials of these reagents.

microbiology↗

bioRxiv2022

The phloem-resident protein OCTOPUS is a novel regulator of flg22-induced responses in Arabidopsis thaliana

Phloem is a critical tissue that transports photosynthates and extracellular signals in vascular plants. Although a functional phloem is necessary for plant health, it is also an ideal environment for pathogens to access host nutrients to promote pathogenesis. Even though many vascular pathogens induce economically relevant crop damage, very little is known about the mechanism(s) by which phloem cells detect potential pathogens and signal to minimize damage. Our lab searched existing phosphoproteomic databases, mining for proteins that were phosphorylated in response to the defense-elicitor flagellin, or flg22, AND were expressed in vascular cells, and we identified Octopus (OPS). OPS is polarly associated with the plasma membrane (PM) of sieve element cells and promotes their differentiation from procambial precursor cells by inhibiting the function of BIN2 in brassinosteroid-related signaling. The observation that OPS is differentially phosphorylated in response to flg22 led us to the examine whether OPS may function in flg22-induced signaling using Arabidopsis T-DNA insertion mutants lacking a functional OPS. In wild-type (WT) seedlings, flg22 binds to the PM receptor flagellin sensing 2 (FLS2) to initiate three branches of a signaling cascade that culminates in increased expression of distinct marker genes. Ultimately these signaling pathways lead to the restriction of pathogen growth. Two independent alleles of ops were treated with 100 M flg22 and marker genes from all three branches of FLS2 signaling exhibited higher expression than WT. We also found that in the absence of any flg22, ops mutants displayed increased flg22 signaling responses. Our results indicate that OPS may function as a negative regulator of flg22-induced signaling events and is one of very few phloem-resident proteins with a documented role in flg22 signaling. These results indicate that the phloem may be able to sense and response to the threat of bacterial pathogens in a unique way.

plant biology↗

bioRxiv2020

Naturally acquired blocking human monoclonal antibodies to Plasmodium vivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolated and characterized anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies blocked PvRBP2b binding to reticulocytes and inhibited complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

microbiology↗

bioRxiv2018

Duffy Antigen Expression in Erythroid Bone Marrow Precursor Cells of Genotypically Duffy Negative Individuals

The gene encoding the Duffy blood group protein (Fy, CD234; additional designations Duffy Antigen Receptor of Chemokines [DARC] and Atypical Chemokine Receptor 1 [ACKR1]) is characterized by a SNP in a GATA-1 transcription factor binding site associated with the erythrocyte silent (ES) phenotype. FYES homozygous people are viewed to be highly resistant to blood stage infection with Plasmodium vivax. Increasingly, however, studies are reporting P. vivax infections in Fy-negative individuals across malarious African countries where FYES approaches genetic fixation. This suggests that P. vivax has evolved a Fy-independent RBC invasion pathway, or that the GATA-1 SNP does not abolish Fy expression. Here, we tested the second hypothesis through binding studies to erythroid lineage cells using recombinant P. vivax Duffy binding protein, the parasites invasion ligand and Fy6-specific antibodies. We first observed variable Fy expression on circulating RBCs, irrespective of FY genotype; FYES RBCs were periodically Fy-positive. Furthermore, during the in vitro erythroid differentiation of CD34+ cells and on ex vivo bone marrow samples, we observed Fy expression on erythroid precursor cells from FYES people. Finally, the Fy6-specific nanobody, CA111 was used to capture Fy from the surface of FYES RBCs. Our findings reveal that the GATA-1 SNP does not fully abolish Fy expression and provide insight on potential susceptibility of Fy-negative people to vivax malaria.\n\nSignificanceDuffy blood group negativity results from a single nucleotide polymorphism (SNP) in the gene promoter, and reaches genetic fixation in many African ethnicities. Because the Duffy protein (Fy) is an important contact point during Plasmodium vivax human red blood cell invasion, Fy-negativity is considered to confer resistance to P. vivax malaria. With recent studies in African countries reporting P. vivax infection in Fy-negative people, we studied Fy expression across erythroid development. Here we report that the FY promoter SNP does not abolish Fy protein expression in erythroid progenitors developing in the bone marrow. These results further emphasizes the importance of reticulocytes as targets for P. vivax blood stage infection and propose a mechanism for P. vivax infections in Fy-negative people.

genetics↗

bioRxiv2018

Human Migration and the Spread of the Nematode Parasite Wuchereria bancrofti

The human disease lymphatic filariasis causes the debilitating effects of elephantiasis and hydrocele. Lymphatic filariasis currently affects the lives of 90 million people in 52 countries. There are three nematodes that cause lymphatic filariasis, Brugia malayi, B. timori, and Wuchereria bancrofti, but 90% of all cases of lymphatic filariasis are caused solely by W. bancrofti. Here we use population genomics to identify the geographic origin of W. bancrofti and reconstruct its spread. Previous genomic sequencing efforts have suffered from difficulties in obtaining Wb DNA. We used selective whole genome amplification to enrich W. bancrofti DNA from infected blood samples and were able to analyze 47 whole genomes of W. bancrofti from endemic locations in Haiti, Mali, Kenya, and Papua New Guinea. Our results are consistent with a Southeast Asia or East Asia origin for W. bancrofti spread around the globe by infecting migrating populations of humans. Austronesians probably introduced W. ban-crofti to Madagascar where later migrations moved it to continental Africa. From Africa, W. bancrofti spread to the New World during the transatlantic slave trade. The greater genetic diversity of W. bancrofti populations from Haiti are also consistent with genetic admixture from multiple source populations. Genome scans for locally adapted haplotypes identified genes associated with human immune suppression and insecticide sensitivity. Locally adapted haplotypes may provide a foundation to understand the distribution of W. bancrofti compared to that of other filarial nematodes and how populations may differ in response to eradication efforts.

evolutionary biology↗