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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Changes in Peripheral Blood Regulatory T Cells and IL-6 and IL-10 Levels Predict Response of Pediatric Medulloblastoma and Germ Cell Tumors With Residual or Disseminated Disease to Craniospinal Irradiation

Radiation therapy (RT) modulates immune cells and cytokines, resulting in both clinically beneficial and detrimental effects. The changes in peripheral blood T lymphocyte subsets and cytokines during RT for pediatric brain tumors and the association of these changes with therapeutic outcomes have not been well described.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2023

Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention

The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an “Achilles heel” for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Organogenetic transcriptomes of the Drosophila embryo at single cell resolution

To gain insight into the transcription programs activated during the formation of Drosophila larval structures, we carried out single cell RNA sequencing during two periods of Drosophila embryogenesis: stages 10-12, when most organs are first specified and initiate morphological and physiological specialization; and stages 13-16, when organs achieve their final mature architectures and begin to function. Our data confirm previous findings with regards to functional specialization of some organs – the salivary gland and trachea – and clarify the embryonic functions of another – the plasmatocytes. We also identify two early developmental trajectories in germ cells and uncover a potential role for proteolysis during germline stem cell specialization. We identify the likely cell type of origin for key components of the Drosophila matrisome and several commonly used Drosophila embryonic cell culture lines. Finally, we compare our findings with other recent related studies and with other modalities for identifying tissue-specific gene expression patterns. These data provide a useful community resource for identifying many new players in tissue-specific morphogenesis and functional specialization of developing organs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Meiotic susceptibility for induction of sperm with chromosomal aberrations in patients receiving combination chemotherapy for Hodgkin lymphoma

Improvements in survival rates with gonad-sparing protocols for childhood and adolescence cancer have increased the optimism of survivors to become parents after treatment. Findings in rodents indicate that chromosomal aberrations can be induced in male germ cells by genotoxic exposures and transmitted to offspring and future generations with effects on development, fertility and health. Thus, there is a need for effective technologies to identify human sperm carrying chromosomal aberrations to assess the germ-line risks, especially for cancer survivors who have received genotoxic therapies. The time-dependent changes in the burden of sperm carrying structural chromosomal aberrations were assessed for the first time in a cancer setting, using the AM8 sperm FISH protocol which simultaneously detects abnormalities in chromosomal structure and number in sperm. Nine Hodgkin lymphoma (HL) patients provided 20 semen samples before, during, and after NOVP therapy (Novantrone, Oncovin, Velban and Prednisone) and radiation therapy that produced scattered gonadal doses from <0.05 to 0.6 Gy. Late meiosis was found to be the most sensitive to NOVP treatment for the production of sperm with chromosomal abnormalities, both in structure and number. Earlier stages of spermatogenesis were less sensitive and there was no evidence that therapy-exposed stem cells resulted in increased frequencies of sperm with abnormalities in chromosomal structure or number. This indicates that NOVP therapy may increase the risks for paternal transmission of chromosomal structural aberrations for sperm produced 32 to 45 days after a treatment with these drugs and implies that there are no excess risks for pregnancies conceived more than 6 months after this therapy. This clinical evaluation of the AM8 sperm FISH protocol indicates that it is a promising tool for assessing an individual’s burden of sperm carrying chromosomal structural aberrations as well as aneuploidies after cancer therapy, with broad applications in other clinical and environmental situations that may pose aneugenic or clastogenic risks to human spermatogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity

In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4 PPH-4.1 phosphatase and ATR ATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4 PPH-4.1 , ATR ATL-1 , and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Dynamic zinc fluxes regulate meiotic progression in Caenorhabditis elegans

Abstract Zinc influx and efflux events are essential for meiotic progression in oocytes of several mammalian and amphibian species, but it is less clear whether this evolutionary conservation of zinc signals is also important in late-stage germline development in invertebrates. Using quantitative, single cell elemental mapping methods, we find that Caenorhabditis elegans oocytes undergo significant stage-dependent fluctuations in total zinc content, rising by over sevenfold from Prophase I through the beginning of mitotic divisions in the embryo. Live imaging of the rapid cell cycle progression in C. elegans enables us to follow changes in labile zinc pools across meiosis and mitosis in single embryo. We find a dynamic increase in labile zinc prior to fertilization that then decreases from Anaphase II through pronuclear fusion and relocalizes to the eggshell. Disruption of these zinc fluxes blocks extrusion of the second polar body, leading to a range of mitotic defects. We conclude that spatial temporal zinc fluxes are necessary for meiotic progression in C. elegans and are a conserved feature of germ cell development in a broad cross section of metazoa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The ICRP, MELODI, and ALLIANCE workshop on effects of ionizing radiation exposure in offspring and next generations: a summary of discussions

Task Group 121 – Effects of ionizing radiation exposure in offspring and next generations – is a task group under the Committee 1 of the International Commission on Radiological Protection (ICRP), approved by the Main Commission on 18th November 2021. The main goals of Task Group 121 are to (1) review and update the scientific literature of relevance to radiation-related effects in the offspring of parent(s) exposed to ionizing radiation in both human and non-human biota; (2) to assess preconceptional and intrauterine effects of radiation exposure and related morbidity and mortality; and, (3) to provide advice about the level of evidence and how to consider these preconceptional and postconceptional effects in the system of radiological protection for humans and non-human biota. The Task Group is reviewing relevant literature since Publication 90 ‘Biological effects after prenatal irradiation (embryo and fetus)’ (2003) and will include radiation-related effects on future generations in humans, animals, and plants. This review will be conducted to account for the health effects on offspring and subsequent generations in the current system of radiological protection. Radiation detriment calculation will also be reviewed. Finally, preliminary recommendations will be made to update the integration of health effects in offspring and next generations in the system of radiological protection. A Workshop, jointly organized by ICRP Task Group 121 and European Radiation Protection Research Platforms MELODI and ALLIANCE was held in Budapest, Hungary, from 31st May to 2nd June 2022. Participants discussed four important topics: (1) hereditary and epigenetic effects due to exposure of the germ cell line (preconceptional exposure), (2) effects arising from exposure of the embryo and fetus (intrauterine exposure), (3) transgenerational effects on biota, and (4) its potential impact on the system of radiological protection. Based on the discussions and presentations during the breakout sessions, newer publications, and gaps on the current scientific literature were identified. For instance, there are some ongoing systematic reviews and radiation epidemiology reviews of intrauterine effects. There are newer methods of Monte Carlo simulation for fetal dosimetry, and advances in radiation genetics, epigenetics, and radiobiology studies. While the current impact of hereditary effects on the global detriment was reported as small, the questions surrounding the effects of radiation exposure on offspring and the next generation are crucial, recurring, and with a major focus on exposed populations. Here, this article summarizes the workshop discussions, presentations, and conclusions of each topic and introduces the special issue of the International Journal of Radiation Biology resulting from the discussions of the meeting.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2022

Zinc dynamics regulate early ovarian follicle development

Zinc fluctuations regulate key steps in late oocyte and pre-implantation embryo development; however, roles for zinc in preceding stages in early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To understand the roles of zinc during early follicle development, we applied single cell X-ray fluorescence microscopy, a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual mouse oocytes and associated somatic cells within early follicles. Here, we report a significant stage-specific increase and compartmental redistribution in oocyte zinc content upon the initiation of early follicle growth. The increase in zinc correlates with the increased expression of specific zinc transporters, including two that are essential in oocyte maturation. While oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, somatic survival and proliferation are significantly more sensitive to zinc chelation or supplementation. Finally, transcriptomic, proteomic, and zinc loading analyses reveal enrichment of zinc targets in the ubiquitination pathway. Overall, these results demonstrate that distinct cell type- specific zinc regulations are required for follicle growth and indicate that physiological fluctuation in the localization and availability of this inorganic cofactor has fundamental functions in early gamete development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas

Highlights: • CAM supports the growth and differentiation of embryonic stem cells into teratomas. • In teratomas, parenchymal Embryonic stem cell-derived endothelial cells form chimeric vessels with stromal endothelial cells. • CAM allows the simultaneously characterization of vasculogenesis and angiogenesis processes. Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression

ABSTRACT Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers’ deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus’s central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS’s function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion. IMPORTANCE Brown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of “omics” platforms has recently uncovered the key “oxidative-hydrolytic” mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes’ catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system’s use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Data for Coprocessing Corn Germ Meal for Oil Recovery and Ethanol Production, a Process Model for Lipid-Producing Energy Crops

Efforts to engineer high-productivity crops to accumulate oils in their vegetative tissue present the possibility of expanding biodiesel production. However, processing the new crops for lipid recovery and ethanol production from cell wall saccharides is challenging and expensive. In a previous study using corn germ meal as a model substrate, we reported that liquid hot water (LHW) pretreatment enriched the lipid concentration by 2.2 to 4.2 fold. This study investigated combining oil recovery with ethanol production by extracting oil following LHW and simultaneous saccharification and co-fermentation (SSCF) of the biomass. Corn germ meal was again used to model the oil-bearing energy crops. Pretreated germ meal hydrolysate or solids (160 °C and 180 °C for 10 minutes) were fermented, and lipids were extracted from both the spent fermentation whole broth and fermentation solids, which were recovered by centrifugation and convective drying. Lipid contents in spent fermentation solids increased 3.7 to 5.7 fold compared to the beginning germ meal. The highest lipid yield achieved after fermentation was 36.0 mg lipid g−1 raw biomass; the maximum relative amount of triacylglycerol (TAG) was 50.9% of extracted oil. Although the fermentation step increased the lipid concentration of the recovered solids, it did not improve the lipid yields of pretreated biomass and detrimentally affected oil compositions by increasing the relative concentrations of free fatty acids.

Conversion↗
DOE OSTI2022

Coprocessing Corn Germ Meal for Oil Recovery and Ethanol Production: A Process Model for Lipid-Producing Energy Crops

Efforts to engineer high-productivity crops to accumulate oils in their vegetative tissue present the possibility of expanding biodiesel production. However, processing the new crops for lipid recovery and ethanol production from cell wall saccharides is challenging and expensive. In a previous study using corn germ meal as a model substrate, we reported that liquid hot water (LHW) pretreatment enriched the lipid concentration by 2.2 to 4.2 fold. This study investigated combining oil recovery with ethanol production by extracting oil following LHW and simultaneous saccharification and co-fermentation (SSCF) of the biomass. Corn germ meal was again used to model the oil-bearing energy crops. Pretreated germ meal hydrolysate or solids (160 and 180 °C for 10 min) were fermented, and lipids were extracted from both the spent fermentation whole broth and fermentation solids, which were recovered by centrifugation and convective drying. Lipid contents in spent fermentation solids increased 3.7 to 5.7 fold compared to the beginning germ meal. The highest lipid yield achieved after fermentation was 36.0 mg lipid g−1 raw biomass; the maximum relative amount of triacylglycerol (TAG) was 50.9% of extracted oil. Although the fermentation step increased the lipid concentration of the recovered solids, it did not improve the lipid yields of pretreated biomass and detrimentally affected oil compositions by increasing the relative concentrations of free fatty acids.

09 BIOMASS FUELS↗
DOE OSTI2025

Sulfoquinovose is exclusively metabolized by the gut microbiota and degraded differently in mice and humans

Abstract Background Sulfoquinovose (SQ) is a green-diet-derived sulfonated glucose and a selective substrate for a limited number of human gut bacteria. Complete anaerobic SQ degradation via interspecies metabolite transfer to sulfonate-respiring bacteria produces hydrogen sulfide, which has dose- and context-dependent health effects. Here, we studied potential SQ degradation by the mammalian host and the impact of SQ supplementation on human and murine gut microbiota diversity and metabolism. Results 13 CO 2 breath tests with germ-free C57BL/6 mice gavaged with 13 C-SQ were negative. Also, SQ was not degraded by human intestinal cells in vitro, indicating that SQ is not directly metabolized by mice and humans. Addition of increasing SQ concentrations to human fecal microcosms revealed dose-dependent responses of the microbiota and corroborated the relevance ofAgathobacter rectalisandBilophila wadsworthiain cooperative degradation of SQ to hydrogen sulfide via interspecies transfer of 2,3-dihydroxy-1-propanesulfonate (DHPS). Similar to the human gut microbiome, the genetic capacity for SQ or DHPS degradation is sparsely distributed among bacterial species in the gut of conventional laboratory mice.Escherichia coliandEnterocloster clostridioformiswere identified as primary SQ degraders in the mouse gut. SQ and DHPS supplementation experiments with conventional laboratory mice and their intestinal contents showed that SQ was incompletely catabolized to DHPS. Although someE. clostridioformisgenomes encode an extended sulfoglycolytic pathway for both SQ and DHPS fermentation, SQ was only degraded to DHPS by a mouse-derivedE. clostridioformisstrain. Conclusions Our findings suggest that SQ is solely a nutrient for the gut microbiota and not for mice and humans, emphasizing its potential as a prebiotic. SQ degradation by the microbiota of conventional laboratory mice differs from the human gut microbiota by absence of DHPS degradation activity. Hence, the microbiota of conventional laboratory mice does not fully represent the SQ metabolism in humans, indicating the need for alternative model systems to assess the impact of SQ on human health. This study advances our understanding of how individual dietary compounds shape the microbial community structure and metabolism in the gut and thereby potentially influence host health.

Microbiology↗
DOE OSTI2021

Attaining the promise of plant gene editing at scale

Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Capsular polysaccharide correlates with immune response to the human gut microbe Ruminococcus gnavus

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

A Germline-Targeting Chimpanzee SIV Envelope Glycoprotein Elicits a New Class of V2-Apex Directed Cross-Neutralizing Antibodies

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germ line-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these animals identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, nonneutralizing antibodies revealed that SCIV-expressed, but not wild-type SIVcpz Envs, as well as a subset of primary HIV-1 Envs, preferentially adopted a more open trimeric state. These results reveal the existence of a cryptic V2 epitope that is exposed in occluded-open SIVcpz and HIV-1 Env trimers and elicits cross-neutralizing responses of limited breadth and potency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Epstein-Barr Virus Epitope–Major Histocompatibility Complex Interaction Combined with Convergent Recombination Drives Selection of Diverse T Cell Receptor α and β Repertoires

Recognition modes of individual T cell receptors (TCRs) are well studied, but factors driving the selection of TCR repertoires from primary through persistent human virus infections are less well understood. Using deep sequencing, we demonstrate a high degree of diversity of Epstein-Barr virus (EBV)-specific clonotypes in acute infectious mononucleosis (AIM). Only 9% of unique clonotypes detected in AIM persisted into convalescence; the majority (91%) of unique clonotypes detected in AIM were not detected in convalescence and were seeming replaced by equally diverse “de novo” clonotypes. The persistent clonotypes had a greater probability of being generated than nonpersistent clonotypes due to convergence recombination of multiple nucleotide sequences to encode the same amino acid sequence, as well as the use of shorter complementarity-determining regions 3 (CDR3s) with fewer nucleotide additions (i.e., sequences closer to germ line). Moreover, the two most immunodominant HLA-A2-restricted EBV epitopes, BRLF1 109 and BMLF1 280 , show highly distinct antigen-specific public (i.e., shared between individuals) features. In fact, TCRα CDR3 motifs played a dominant role, while TCRβ played a minimal role, in the selection of TCR repertoire to an immunodominant EBV epitope, BRLF1. This contrasts with the majority of previously reported repertoires, which appear to be selected either on TCRβ CDR3 interactions with peptide/major histocompatibility complex (MHC) or in combination with TCRα CDR3. Understanding of how TCR-peptide-MHC complex interactions drive repertoire selection can be used to develop optimal strategies for vaccine design or generation of appropriate adoptive immunotherapies for viral infections in transplant settings or for cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISS OMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded β-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISS OMP . The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a β-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISS OMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

59 BASIC BIOLOGICAL SCIENCES↗