Post-translational modification directs nuclear and hyphal tip localization of Candida albicans mRNA-binding protein Slr1

The morphological transition of the opportunistic fungal pathogen Candida albicans from budding to hyphal growth has been implicated in its ability to cause disease in animal models. Absence of SR-like RNA-binding protein Slr1 slows hyphal formation and decreases virulence in a systemic candidiasis model, suggesting a role for post-transcriptional regulation in these processes. SR (serine–arginine)-rich proteins influence multiple steps in mRNA metabolism and their localization and function are frequently controlled by modification. We now demonstrate that Slr1 binds to polyadenylated RNA and that its intracellular localization is modulated by phosphorylation and methylation. Wildtype Slr1-GFP is predominantly nuclear, but also co-fractionates with translating ribosomes. The non-phosphorylatable slr1-6SA-GFP protein, in which six serines in SR/RS clusters are substituted with alanines, primarily localizes to the cytoplasm in budding cells. Intriguingly, hyphal cells display a slr1-6SA-GFP focus at the tip near the Spitzenkörper, a vesicular structure involved in molecular trafficking to the tip. The presence of slr1-6SA-GFP hyphal tip foci is reduced in the absence of the mRNA-transport protein She3, suggesting that unphosphorylated Slr1 associates with mRNA–protein complexes transported to the tip. The impact of SLR1 deletion on hyphal formation and function thus may be partially due to a role in hyphal mRNA transport

Structure and mechanism of monoclonal antibody binding to the junctional epitope of Plasmodium falciparum circumsporozoite protein.

Lasting protection has long been a goal for malaria vaccines. The major surface antigen on Plasmodium falciparum sporozoites, the circumsporozoite protein (PfCSP), has been an attractive target for vaccine development and most protective antibodies studied to date interact with the central NANP repeat region of PfCSP. However, it remains unclear what structural and functional characteristics correlate with better protection by one antibody over another. Binding to the junctional region between the N-terminal domain and central NANP repeats has been proposed to result in superior protection: this region initiates with the only NPDP sequence followed immediately by NANP. Here, we isolated antibodies in Kymab mice immunized with full-length recombinant PfCSP and two protective antibodies were selected for further study with reactivity against the junctional region. X-ray and EM structures of two monoclonal antibodies, mAb667 and mAb668, shed light on their differential affinity and specificity for the junctional region. Importantly, these antibodies also bind to the NANP repeat region with equal or better affinity. A comparison with an NANP-only binding antibody (mAb317) revealed roughly similar but statistically distinct levels of protection against sporozoite challenge in mouse liver burden models, suggesting that junctional antibody protection might relate to the ability to also cross-react with the NANP repeat region. Our findings indicate that additional efforts are necessary to isolate a true junctional antibody with no or much reduced affinity to the NANP region to elucidate the role of the junctional epitope in protection

Modeling the differentiation of induced pluripotent stem cells using single cell RNA sequencing data

Induced pluripotent stem cells (iPSCs) are promising tools for biomedical research such as human disease model, and regenerative medicine. Yet, our insight into the gene regulatory mechanism of iPSC differentiation is still incomplete. This study aims to understand the regulatory mechanism underlying the iPSC differentiation via a novel combined approach that utilizes both scRNA-seq data analysis and network modeling. In this study, the scRNA-seq data measure the gene expression of iPSCs differentiating into four different cell types: mesoderms, endoderms, neurons, and trophectoderms (TEs), and at four collection times: day 1,2,4, and 6. Based on hierarchical clustering analysis, we first found the 640 embryonic developmental genes can capture major cell phenotypes. The results also show that after iPSCs differentiate into TEs, around day 4 and 6 the TEs diverge into two subpopulations. In subsequent analyses, we inferred a pseudotime of each cell and examined the expression dynamics of the genes in the TE cell line and clustered the genes based on their dynamical behaviors. Literature information and gene expression dynamics were used to construct a transcription factor network, and the network model reveals five steady states, which can explain the mechanism of the TE cell differentiation

Structural and biophysical approaches to the understanding of immunity and host-pathogen interactions in human malaria

Malaria remains a global health challenge with significant morbidity and mortality. This thesis describes host-pathogen interactions in two malaria parasites with the greatest human impact, Plasmodium falciparum and Plasmodium vivax. I aimed to shed light on the fundamental biology of antibody responses against P. falciparum and the invasion of erythrocytes by P. vivax, offering potential insights into design and development of novel vaccines and antibody therapeutics. Previous efforts over many years in P. falciparum vaccine development led to the recent approval of the RTS,S/AS01 vaccine, which is a recombinant vaccine based on P. falciparum circumsporozoite protein (PfCSP). This vaccine, while a milestone, has certain limitations in terms of efficacy and durability of the antibodies. My thesis research has provided structural insights into the antibody responses against the CSP. Crystal structures of several diverse antibody Fabs targeting the central NANP repeat region of CSP reveal dominant usage of germline-encoded aromatic residues to bind to their repeat paratopes, which frequently comprise conserved secondary structural motifs formed by the NPNA sequence. The structures also reinforce the importance of homotypic Fab-Fab interactions among anti-NANP antibodies that lead to an increase in affinity due to an avidity effect. These findings support the hypothesis that PfCSP may function as an immune decoy, potentially limiting the maturation of B-cell responses and may explain non-durable antibody responses seen in the current vaccines. Additionally, this study identifies antibodies targeting the less-explored N- and C-terminal domains of PfCSP that inhibit parasite infection in vivo, suggesting a potential role of these two domains in protective humoral immunity against P. falciparum. The characterization of these antibodies also reveals a new, highly conserved C-terminal epitope, representing a potential strain-transcending target that could be incorporated into future vaccines. The second part of this thesis explores P. vivax Duffy-binding protein (PvDBP), an essential component in the invasion of human red blood cells, binds to its receptor, DARC. Sulfation of tyrosine residues on DARC affects its binding to PvDBP, but this interaction with sulfated tyrosine has not been visualized as previous studies only show a small, non-sulfated, helical peptide from DARC binding to region II of PvDBP (PvDBP-RII). Here, the structure of PvDBP-RII bound to sulfated DARC peptide shows a sulfate on Y41 binds to a positively charged pocket on PvDBP-RII. Affinity measurement shows that sulfated Y41 on DARC is crucial to high-affinity binding to PvDBP-RII, and the importance of tyrosine sulfation is also confirmed in growth-inhibition experiments in parasites. This thesis also introduces a promising vaccine candidate based on a conserved subdomain of PvDBP. This candidate, termed ‘Interface,’ demonstrates superior thermal stability and immunogenicity compared to the existing construct, holding promise for large-scale production and distribution in malaria-endemic regions. Overall, this thesis advances our understanding of the immune responses against these promising vaccine candidates and the biology of P. vivax invasion and paves the way for the development of next-generation malaria vaccines and antibody therapeutics

Structural basis for DARC binding in reticulocyte invasion by Plasmodium vivax.

The symptoms of malaria occur during the blood stage of infection, when the parasite replicates within human red blood cells. The human malaria parasite, Plasmodium vivax, selectively invades reticulocytes in a process which requires an interaction between the ectodomain of the human DARC receptor and the Plasmodium vivax Duffy-binding protein, PvDBP. Previous studies have revealed that a small helical peptide from DARC binds to region II of PvDBP (PvDBP-RII). However, it is also known that sulphation of tyrosine residues on DARC affects its binding to PvDBP and these residues were not observed in previous structures. We therefore present the structure of PvDBP-RII bound to sulphated DARC peptide, showing that a sulphate on tyrosine 41 binds to a charged pocket on PvDBP-RII. We use molecular dynamics simulations, affinity measurements and growth-inhibition experiments in parasites to confirm the importance of this interaction. We also reveal the epitope for vaccine-elicited growth-inhibitory antibody DB1. This provides a complete understanding of the binding of PvDBP-RII to DARC and will guide the design of vaccines and therapeutics to target this essential interaction

Structural basis for DARC binding in reticulocyte invasion by Plasmodium vivax

Abstract The symptoms of malaria occur during the blood stage of infection, when the parasite replicates within human red blood cells. The human malaria parasite, Plasmodium vivax, selectively invades reticulocytes in a process which requires an interaction between the ectodomain of the human DARC receptor and the Plasmodium vivax Duffy-binding protein, PvDBP. Previous studies have revealed that a small helical peptide from DARC binds to region II of PvDBP (PvDBP-RII). However, it is also known that sulphation of tyrosine residues on DARC affects its binding to PvDBP and these residues were not observed in previous structures. We therefore present the structure of PvDBP-RII bound to sulphated DARC peptide, showing that a sulphate on tyrosine 41 binds to a charged pocket on PvDBP-RII. We use molecular dynamics simulations, affinity measurements and growth-inhibition experiments in parasites to confirm the importance of this interaction. We also reveal the epitope for vaccine-elicited growth-inhibitory antibody DB1. This provides a complete understanding of the binding of PvDBP-RII to DARC and will guide the design of vaccines and therapeutics to target this essential interaction

A cross-neutralizing antibody between HIV-1 and influenza virus

Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.</p

Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection

Abstract The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family

A novel CSP C-terminal epitope targeted by an antibody with protective activity against Plasmodium falciparum.

Potent and durable vaccine responses will be required for control of malaria caused by Plasmodium falciparum (Pf). RTS,S/AS01 is the first, and to date, the only vaccine that has demonstrated significant reduction of clinical and severe malaria in endemic cohorts in Phase 3 trials. Although the vaccine is protective, efficacy declines over time with kinetics paralleling the decline in antibody responses to the Pf circumsporozoite protein (PfCSP). Although most attention has focused on antibodies to repeat motifs on PfCSP, antibodies to other regions may play a role in protection. Here, we expressed and characterized seven monoclonal antibodies to the C-terminal domain of CSP (ctCSP) from volunteers immunized with RTS,S/AS01. Competition and crystal structure studies indicated that the antibodies target two different sites on opposite faces of ctCSP. One site contains a polymorphic region (denoted α-ctCSP) and has been previously characterized, whereas the second is a previously undescribed site on the conserved β-sheet face of the ctCSP (denoted β-ctCSP). Antibodies to the β-ctCSP site exhibited broad reactivity with a diverse panel of ctCSP peptides whose sequences were derived from field isolates of P. falciparum whereas antibodies to the α-ctCSP site showed very limited cross reactivity. Importantly, an antibody to the β-site demonstrated inhibition activity against malaria infection in a murine model. This study identifies a previously unidentified conserved epitope on CSP that could be targeted by prophylactic antibodies and exploited in structure-based vaccine design