Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

Subcellular proteomics for determining iron-limited remodeling of plastids in the model diatom Thalassiosira pseudonana (Bacillariophyta).

Diatoms are important primary producers in the world's oceans, yet their growth is constrained in large regions by low bioavailable iron (Fe). Low-Fe stress-induced limitation of primary production is due to requirements for Fe in components of essential metabolic pathways including photosynthesis and other chloroplast plastid functions. Studies have shown that under low-Fe stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and in protein abundances within the diatom plastid. While in silico predictions provide putative information on plastid-localized proteins, knowledge of diatom plastid proteins remains limited in comparison to well-studied model photosynthetic organisms. To address this, we employed shotgun proteomics to investigate the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana to gain a better understanding of how the plastid proteome is remodeled in response to Fe limitation. Using mass spectrometry-based peptide identification and quantification, we analyzed T. pseudonana grown under Fe-replete and -limiting conditions. Through these analyses, we inferred the relative quantities of each protein, revealing that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle. Additionally, we observed changes in the expression of light-harvesting proteins. In silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical versus observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.

Oligotrophic waters of the Northwest Atlantic support taxonomically diverse diatom communities that are distinct from coastal waters.

Diatoms are important components of the marine food web and one of the most species-rich groups of phytoplankton. The diversity and composition of diatoms in eutrophic nearshore habitats have been well documented due to the outsized influence of diatoms on coastal ecosystem functioning. In contrast, patterns of both diatom diversity and community composition in offshore oligotrophic regions where diatom biomass is low have been poorly resolved. To compare the diatom diversity and community composition in oligotrophic and eutrophic waters, diatom communities were sampled along a 1,250 km transect from the oligotrophic Sargasso Sea to the coastal waters of the northeast US shelf. Diatom community composition was determined by amplifying and sequencing the 18S rDNA V4 region. Of the 301 amplicon sequence variants (ASVs) identified along the transect, the majority (70%) were sampled exclusively from oligotrophic waters of the Gulf Stream and Sargasso Sea and included the genera Bacteriastrum, Haslea, Hemiaulus, Pseudo-nitzschia, and Nitzschia. Diatom ASV richness did not vary along the transect, indicating that the oligotrophic Sargasso Sea and Gulf Stream are occupied by a diverse diatom community. Although ASV richness was similar between oligotrophic and coastal waters, diatom community composition in these regions differed significantly and was correlated with temperature and phosphate, two environmental variables known to influence diatom metabolism and geographic distribution. In sum, oligotrophic waters of the western North Atlantic harbor diverse diatom assemblages that are distinct from coastal regions, and these open ocean diatoms warrant additional study, as they may play critical roles in oligotrophic ecosystems.

Plasmodium falciparum in Aotus nancymaae: A New Model for Placental Malaria.

Plasmodium falciparum-infected erythrocytes that display the variant surface antigen VAR2CSA bind chondroitin sulfate A (CSA) to sequester in placental intervillous spaces, causing severe sequelae for mother and offspring. Here, we establish a placental malaria (PM) monkey model. Pregnant Aotus infected with CSA-binding P. falciparum CS2 parasites during the third trimester developed pronounced sequestration of late-stage parasites in placental intervillous spaces that express VAR2CSA and bind specifically to CSA. Similar to immune multigravid women, a monkey infected with P. falciparum CS2 parasites over successive pregnancies acquired antibodies against VAR2CSA, with potent functional activity that was boosted upon subsequent pregnancy infections. Aotus also developed functional antibodies after multiple acute PM episodes and subsequent VAR2CSA immunization. In summary, P. falciparum infections in pregnant Aotus monkeys recapitulate all the prominent features of human PM infection and immunity, and this model can be useful for basic mechanistic studies and preclinical studies to qualify candidate PM vaccines. Clinical Trials Registration: NCT02471378.

An Invariant Protein That Colocalizes With VAR2CSA on Plasmodium falciparum-Infected Red Cells Binds to Chondroitin Sulfate A.

BACKGROUND: Plasmodium falciparum-infected red blood cells (iRBCs) bind and sequester in deep vascular beds, causing malaria-related disease and death. In pregnant women, VAR2CSA binds to chondroitin sulfate A (CSA) and mediates placental sequestration, making it the major placental malaria (PM) vaccine target. METHODS: In this study, we characterize an invariant protein associated with PM called P falciparum chondroitin sulfate A ligand (PfCSA-L). RESULTS: Recombinant PfCSA-L binds both placental CSA and VAR2CSA with nanomolar affinity, and it is coexpressed on the iRBC surface with VAR2CSA. Unlike VAR2CSA, which is anchored by a transmembrane domain, PfCSA-L is peripherally associated with the outer surface of knobs through high-affinity protein-protein interactions with VAR2CSA. This suggests that iRBC sequestration involves complexes of invariant and variant surface proteins, allowing parasites to maintain both diversity and function at the iRBC surface. CONCLUSIONS: The PfCSA-L is a promising target for intervention because it is well conserved, exposed on infected cells, and expressed and localized with VAR2CSA.

Emerging harmful algal blooms caused by distinct seasonal assemblages of a toxic diatom.

Diatoms in the Pseudo-nitzschia genus produce the neurotoxin domoic acid. Domoic acid bioaccumulates in shellfish, causing illness in humans and marine animals upon ingestion. In 2017, high domoic acid levels in shellfish meat closed shellfish harvest in Narragansett Bay, Rhode Island for the first and only time in history, although abundant Pseudo-nitzschia have been observed for over 60 years. To investigate whether an environmental factor altered endemic Pseudo-nitzschia physiology or new domoic acid-producing strain(s) were introduced to Narragansett Bay, we conducted weekly sampling from 2017 to 2019 and compared closure samples. Plankton-associated domoic acid was quantified by LC-MS/MS and Pseudo-nitzschia spp. were identified using a taxonomically improved high-throughput rDNA sequencing approach. Comparison with environmental data revealed a detailed understanding of domoic acid dynamics and seasonal multi-species assemblages. Plankton-associated domoic acid was low throughout 2017-2019, but recurred in fall and early summer maxima. Fall domoic acid maxima contained known toxic species as well as a novel Pseudo-nitzschia genotype. Summer domoic acid maxima included fewer species but also known toxin producers. Most 2017 closure samples contained the particularly concerning toxic species, P. australis, which also appeared infrequently during 2017-2019. Recurring Pseudo-nitzschia assemblages were driven by seasonal temperature changes, and plankton-associated domoic acid correlated with low dissolved inorganic nitrogen. Thus, the Narragansett Bay closures were likely caused by both resident assemblages that become toxic depending on nutrient status as well as the episodic introductions of toxic species from oceanographic and climatic shifts.

Metatranscriptome analyses indicate resource partitioning between diatoms in the field.

Diverse communities of marine phytoplankton carry out half of global primary production. The vast diversity of the phytoplankton has long perplexed ecologists because these organisms coexist in an isotropic environment while competing for the same basic resources (e.g., inorganic nutrients). Differential niche partitioning of resources is one hypothesis to explain this "paradox of the plankton," but it is difficult to quantify and track variation in phytoplankton metabolism in situ. Here, we use quantitative metatranscriptome analyses to examine pathways of nitrogen (N) and phosphorus (P) metabolism in diatoms that cooccur regularly in an estuary on the east coast of the United States (Narragansett Bay). Expression of known N and P metabolic pathways varied between diatoms, indicating apparent differences in resource utilization capacity that may prevent direct competition. Nutrient amendment incubations skewed N/P ratios, elucidating nutrient-responsive patterns of expression and facilitating a quantitative comparison between diatoms. The resource-responsive (RR) gene sets deviated in composition from the metabolic profile of the organism, being enriched in genes associated with N and P metabolism. Expression of the RR gene set varied over time and differed significantly between diatoms, resulting in opposite transcriptional responses to the same environment. Apparent differences in metabolic capacity and the expression of that capacity in the environment suggest that diatom-specific resource partitioning was occurring in Narragansett Bay. This high-resolution approach highlights the molecular underpinnings of diatom resource utilization and how cooccurring diatoms adjust their cellular physiology to partition their niche space.

The Phaeodactylum genome reveals the evolutionary history of diatom genomes.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes ( approximately 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.

Temporal Dynamics of Cyanobacterial Bloom Community Composition and Toxin Production from Urban Lakes.

With a long evolutionary history and a need to adapt to a changing environment, cyanobacteria in freshwater systems use specialized metabolites for communication, defense, and physiological processes. However, the role that these metabolites play in differentiating species, maintaining microbial communities, and generating niche persistence and expansion is poorly understood. Furthermore, many cyanobacterial specialized metabolites and toxins present significant human health concerns due to their liver toxicity and their potential impact to drinking water. Gaps in knowledge exist with respect to changes in species diversity and toxin production during a cyanobacterial bloom (cyanoHAB) event; addressing these gaps will improve understanding of impacts to public and ecological health. In the current project, we utilized a multiomics strategy (DNA metabarcoding and metabolomics) to determine the cyanobacterial community composition, toxin profile, and the specialized metabolite pool at three freshwater lakes in Providence, RI during summer-fall cyanoHABs. Species diversity decreased at all study sites over the course of the bloom event, and toxin production reached a maximum at the midpoint of the event. Additionally, LC-MS/MS-based molecular networking identified new toxin congeners. This work provokes intriguing questions with respect to the use of allelopathy by organisms in these systems and the presence of emerging toxic compounds that can impact public health.

Simulated upwelling and marine heatwave events promote similar growth rates but differential domoic acid toxicity in Pseudo-nitzschia australis.

Along the west coast of the United States, highly toxic Pseudo-nitzschia blooms have been associated with two contrasting regional phenomena: seasonal upwelling and marine heatwaves. While upwelling delivers cool water rich in pCO2 and an abundance of macronutrients to the upper water column, marine heatwaves instead lead to warmer surface waters, low pCO2, and reduced nutrient availability. Understanding Pseudo-nitzschia dynamics under these two conditions is important for bloom forecasting and coastal management, yet the mechanisms driving toxic bloom formation during contrasting upwelling vs. heatwave conditions remain poorly understood. To gain a better understanding of what drives Pseudo-nitzschia australis growth and toxicity during these events, multiple-driver scenario or 'cluster' experiments were conducted using temperature, pCO2, and nutrient levels reflecting conditions during upwelling (13 °C, 900 ppm pCO2, replete nutrients) and two intensities of marine heatwaves (19 °C or 20.5 °C, 250 ppm pCO2, reduced macronutrients). While P. australis grew equally well under both heatwave and upwelling conditions, similar to what has been observed in the natural environment, cells were only toxic in the upwelling treatment. We also conducted single-driver experiments to gain a mechanistic understanding of which drivers most impact P. australis growth and toxicity. These experiments indicated that nitrogen concentration and N:P ratio were likely the drivers that most influenced domoic acid production, while the impacts of temperature or pCO2 concentration were less pronounced. Together, these experiments may help to provide both mechanistic and holistic perspectives on toxic P. australis blooms in the dynamic and changing coastal ocean, where cells interact simultaneously with multiple altered environmental variables.

A single full-length VAR2CSA ectodomain variant purifies broadly neutralizing antibodies against placental malaria isolates.

Placental malaria (PM) is a deadly syndrome most frequent and severe in first pregnancies. PM results from accumulation of Plasmodium falciparum-infected erythrocytes (IE) that express the surface antigen VAR2CSA and bind to chondroitin sulfate A (CSA) in the placenta. Women become PM-resistant over successive pregnancies as they develop anti-adhesion and anti-VAR2CSA antibodies, supporting VAR2CSA as the leading PM-vaccine candidate. However, the first VAR2CSA subunit vaccines failed to induce broadly neutralizing antibody and it is known that naturally acquired antibodies target both variant-specific and conserved epitopes. It is crucial to determine whether effective vaccines will require incorporation of many or only a single VAR2CSA variants. Here, IgG from multigravidae was sequentially purified on five full-length VAR2CSA ectodomain variants, thereby depleting IgG reactivity to each. The five VAR2CSA variants purified ~0.7% of total IgG and yielded both strain-transcending and strain-specific reactivity to VAR2CSA and IE-surface antigen. In two independent antibody purification/depletion experiments with permutated order of VAR2CSA variants, IgG purified on the first VAR2CSA antigen displayed broad cross-reactivity to both recombinant and native VAR2CSA variants, and inhibited binding of all isolates to CSA. IgG remaining after depletion on all variants showed significantly reduced binding-inhibition activity compared to initial total IgG. These findings demonstrate that a single VAR2CSA ectodomain variant displays conserved epitopes that are targeted by neutralizing (or binding-inhibitory) antibodies shared by multiple parasite strains, including maternal isolates. This suggests that a broadly effective PM-vaccine can be achieved with a limited number of VAR2CSA variants.

Contracting malaria during pregnancy ­ especially a first pregnancy ­ can lead to a severe, placental form of the disease that is often fatal. Red blood cells infected with the malaria parasite Plasmodium falciparum display a protein, VAR2CSA, which can recognize and bind CSA molecules present on placental cells and in placental blood spaces. This leads to the infected blood cells accumulating in the placenta and inducing harmful inflammation. Having been exposed to the parasite in prior pregnancies generates antibodies that target VAR2CSA, stopping the infected blood cells from latching onto placental CSA or tagging them for immune destruction. Overall, this makes placental malaria less severe in following pregnancies, and suggests that vaccines could be developed based on VAR2CSA. However, this protein has regions that can vary in structure, meaning that P. falciparaum can generate many VAR2CSA variants. Individuals exposed to the parasite naturally generate antibodies that block a wide array of variants from attaching to CSA. In contrast, first-generation vaccines based on VAR2CSA fragments have only induced variant-specific antibodies, therefore offering limited protection against infection. As a response, Doritchamou et al. set out to find VAR2CSA structures that could be recognized by antibodies targeting an array of variants. Blood was obtained from women who had had multiple pregnancies and were immune to malaria. Their plasma was passed over five different large VAR2CSA variants in order to isolate and purify antibodies that attached to these structures. Doritchamou et al. found that antibodies binding to individual VAR2CSA structures could also recognise a wide array of VAR2CSA variants and blocked all tested parasites from sticking to CSA. While further research is needed, these findings highlight antibodies that cross-react to diverse VAR2CSA variants and could be used to design more effective vaccines targeting placental malaria.

Draft Genome Sequence of Salegentibacter sp. Strain BDJ18, a Plankton-Associated Bacterium in the Northeast Atlantic Ocean.

Salegentibacter sp. strain BDJ18 was isolated from a plankton-associated seawater sample from the northeast Atlantic Ocean. We report its draft genome assembly, which includes genes potentially important for microbial interactions in the marine environment.

A human monoclonal antibody blocks malaria transmission and defines a highly conserved neutralizing epitope on gametes.

Malaria elimination requires tools that interrupt parasite transmission. Here, we characterize B cell receptor responses among Malian adults vaccinated against the first domain of the cysteine-rich 230 kDa gamete surface protein Pfs230, a key protein in sexual stage development of P. falciparum parasites. Among nine Pfs230 human monoclonal antibodies (mAbs) that we generated, one potently blocks transmission to mosquitoes in a complement-dependent manner and reacts to the gamete surface; the other eight show only low or no blocking activity. The structure of the transmission-blocking mAb in complex with vaccine antigen reveals a large discontinuous conformational epitope, specific to domain 1 of Pfs230 and comprising six structural elements in the protein. The epitope is conserved, suggesting the transmission-blocking mAb is broadly functional. This study provides a rational basis to improve malaria vaccines and develop therapeutic antibodies for malaria elimination.

Empirical bayes analysis of sequencing-based transcriptional profiling without replicates.

BACKGROUND: Recent technological advancements have made high throughput sequencing an increasingly popular approach for transcriptome analysis. Advantages of sequencing-based transcriptional profiling over microarrays have been reported, including lower technical variability. However, advances in technology do not remove biological variation between replicates and this variation is often neglected in many analyses. RESULTS: We propose an empirical Bayes method, titled Analysis of Sequence Counts (ASC), to detect differential expression based on sequencing technology. ASC borrows information across sequences to establish prior distribution of sample variation, so that biological variation can be accounted for even when replicates are not available. Compared to current approaches that simply tests for equality of proportions in two samples, ASC is less biased towards highly expressed sequences and can identify more genes with a greater log fold change at lower overall abundance. CONCLUSIONS: ASC unifies the biological and statistical significance of differential expression by estimating the posterior mean of log fold change and estimating false discovery rates based on the posterior mean. The implementation in R is available at http://www.stat.brown.edu/Zwu/research.aspx.

Phylogenetic diversity of cyanobacterial narB genes from various marine habitats.

Nitrate, the most abundant combined, dissolved form of inorganic nitrogen in global oceans, is a common source of nitrogen (N) for phytoplankton including cyanobacteria. Using a nested polymerase chain reaction (PCR) method, the diversity of the cyanobacterial nitrate reductase gene, narB, was examined in plankton samples from a variety of marine habitats. A total of 480 narB gene fragment sequences were obtained from a coastal coral reef (Heron Island, Australia), open-ocean tropical and subtropical oceanic waters (Atlantic and Pacific Oceans) and a temperate N. Pacific Ocean site (34 degrees N, 129 degrees W). Phylogenetic analyses distinguished eight picocyanobacterial narB clades comprised of DNA sequences derived from the nutrient-replete coastal, nutrient-deplete pelagic and tidally influenced coral reef habitats. The phylogeny of recovered narB gene sequences was consistent with 16S rRNA and ITS sequence phylogenies, suggesting minimal horizontal gene transfer of the narB gene. Depending on sampled habitat, environmental narB sequence types segregated into three divisions: non-picocyanobacterial, coastal picocyanobacterial and open-ocean picocyanobacterial sequences. Using a reverse transcription PCR method, narB mRNA sequences were amplified from Heron Island samples, indicating that narB expression can be detected in environmental samples.

Characterization of cyanobacterial glnA gene diversity and gene expression in marine environments.

PCR primers were designed and used to amplify glnA, the gene that encodes glutamine synthetase, from pure cultures of cyanobacteria and four samples from different marine environments. The glnA phylogeny was similar to that of the 16S rRNA gene, indicating that glnA gene sequences can be used to identify cyanobacteria expressing the glnA gene. Diverse unicellular cyanobacteria glnA genes were recovered from the North Pacific Subtropical Gyre, Monterey Bay, Chesapeake Bay and waters off the New Jersey coast. The majority of sequences were closely related to sequences from Synechococcus strains (78-88% identical DNA sequences). A few sequences that clustered with Prochlorococcus glnA genes were recovered from Monterey Bay and the North Pacific Subtropical Gyre. The expression of glnA was assayed by reverse transcriptase PCR to determine if there was a daily pattern in gene expression of samples collected from New Jersey's Longterm Environmental Observatory site (LEO-15). glnA expression varied over the day, with different glnA sequence types exhibiting different daily cycles. Results showed that the glnA gene can be used to characterize the diversity of natural populations of cyanobacteria, and to characterize gene expression patterns of individual species or strains.

Preferential depletion of zinc within Costa Rica upwelling dome creates conditions for zinc co-limitation of primary production.

The Costa Rica Dome (CRD) is a wind-driven feature characterized by high primary production and an unusual cyanobacterial bloom in surface waters. It is not clear whether this bloom arises from top-down or bottom-up processes. Several studies have argued that trace metal geochemistry within the CRD contributes to the composition of the phytoplankton assemblages, since cyanobacteria and eukaryotic phytoplankton have different transition metal requirements. Here, we report that total dissolved zinc (Zn) is significantly depleted relative to phosphate (P) and silicate (Si) within the upper water column of the CRD compared with other oceanic systems, and this may create conditions favorable for cyanobacteria, which have lower Zn requirements than their eukaryotic competitors. Shipboard grow-out experiments revealed that while Si was a limiting factor under our experimental conditions, additions of Si and either iron (Fe) or Zn led to higher biomass than Si additions alone. The addition of Fe and Zn alone did not lead to significant enhancements. Our results suggest that the depletion of Zn relative to P in upwelled waters may create conditions in the near-surface waters that favor phytoplankton with low Zn requirements, including cyanobacteria.

Characterization of a Plasmodium falciparum Orthologue of the Yeast Ubiquinone-Binding Protein, Coq10p.

Coenzyme Q (CoQ, ubiquinone) is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10), which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.