Biology subjects

Lee, K. K.

Publications and source records attributed to Lee, K. K..

At least 19 recordsLinked to original sources

bioRxiv2026

The ecological context of enzymatic variation

Among the challenges in understanding microbial ecosystems is the presence of physics at vastly different scales. Reactions, catalyzed by enzymes but regulated at the level of the cell, propel the flux of carbon and nitrogen through our atmosphere. Compounding this is the presence of pervasive enzyme sequence variation; this variation has been shown to contain coevolving modes of amino acids which encode the enzyme's evolutionary history. In this work, we take steps towards bridging the gap between the enzymatic variation present in an ecosystem and its subsequent activity. We employ the reduction of nitrate by NarG as a model system, which acts as an essential step in the nitrogen cycle by mediating both the return of di-nitrogen to the atmosphere and the assimilation of nitrate into biomass. Considering both metagenomic reconstructions as well as functional data from soil nitrate reducers, we find that sequence variants of enzymes obey predictable responses to environmental fluctuations. That is, while prior community-level metagenomic studies have characterized the response of bacterial strains to the environment, our study provides an enzyme variant level sequence-to-response map. Further, we demonstrate that a simple statistical model can predict the organismal phenotype from variant sequence; in soil samples not originally seen by that model, the prediction of a variant's reduction rate correlates with how much cells with that variant grow in abundance.

biophysics↗

bioRxiv2026

Cryo-EM mapping of circumsporozoite protein epitope accessibility and visualization of antibody-induced shedding on Plasmodium falciparum sporozoites

The circumsporozoite protein (CSP) on the surface of the sporozoite stage of Plasmodium falciparum parasites is one of the major malaria antigens recognized by the adaptive immune system. Antibodies against CSP can provide protection from malaria infection following mosquito bite of the human host. Recent studies have shown how such antibodies recognize recombinant, truncated forms of Plasmodium falciparum CSP (PfCSP); however, much remains unknown about epitope accessibility and presentation of PfCSP on the parasite itself. Understanding the nature of the native antigen is of significant interest for optimization of immunogen design, since immunization with whole attenuated sporozoites has been shown to provide long-lasting protection against malaria disease. Here we use cryo-electron microscopy and tomography to investigate how antibodies against various PfCSP epitopes (N-terminal domain (NTD), Junction, central repeat, and C-terminal domain (CTD)) impact the PfCSP coat on whole Plasmodium falciparum sporozoites (PfSPZ). We observe that PfCSP by itself cannot be visualized on the surface of the cell by cryo-EM. However, by titrating in antibodies that recognize the Junction motif and central repeats, we delineate a boundary layer formed by PfCSP on the sporozoite outer membrane. The antibody concentration series also enabled us to determine conditions required to induce the dramatic CSP shedding reaction. By comparing matched monovalent FAb with IgG, we demonstrate that antibody bivalency is critical to driving this reaction. Additionally, we demonstrate that even at high concentrations, antibody against the NTD does not bind, indicating the absence or inaccessibility of this epitope on the PfCSP displayed by radiation-attenuated sporozoites, while the CTD can be bound by antibodies without producing a CSP shedding reaction. These studies shed light on the nature of the PfCSP coat in its native context on the parasite, thus revealing the sporozoites antigenic profile.

microbiology↗

bioRxiv2026

Parallelised detection of bacteria viability using an electrode array and the Exeter Multiscope

Antimicrobial resistance remains a global existential threat. Given that antimicrobial therapy commonly starts before pathogen identification, rapid and scalable methods capable of determining effective antimicrobial compounds are needed. In this paper, we demonstrate a 2 x 2 array of parallelised microscopes that uses low numerical aperture (NA=0.25) detection optics and LED excitation to determine bacterial viability based on their fluorescence response to an electrical stimulus. Following a 2-hour incubation, the fluorescent viability readout requires less than one minute. We use K-means clustering to classify pixels in a time lapse sequence of widefield fluorescence images and extract changes seen within bacterial clusters. We demonstrate sufficient sensitivity to measure fluorescence changes after electrical stimulation in a bacterial monolayer. To capture these subtle fluorescence changes at high signal-to-background ratios, we place a limit on the minimum optical density of the bacterial sample. This novel approach is scalable to 96-well formats using a suitable consumable electrode array.

microbiology↗

bioRxiv2026

Reconstructing a Missing Link of HIV-1 Assembly: HIV-1 Envelope-Matrix Interactions in a Native Viral Context

The envelope surface glycoprotein (Env) on HIV-1 drives cell entry and genome delivery through its receptor binding and membrane fusion activities. Incorporation of Env onto assembling virions is governed by its cytoplasmic tail (Env-CT) and the matrix (MA) domain of the PR55Gag (Gag) polyprotein. To better understand how Gag recruits Env onto virions while reducing its antigenic profile by restricting Env copy number to low levels, we used cryo-electron tomography (cryo-ET) and subtomogram averaging combined with molecular dynamics simulations to investigate Env-MA association in intact viral particles. Full-length Env-CT was resolved directly over individual MA trimers in regions of MA lattice discontinuity. We observed that the conserved but enigmatic Kennedy-sequence motif in Env-CT forms a key linkage between Env and MA. Mutational analysis confirmed the impact of specific CT and MA interactions on Env incorporation. Gag maturation released MA lattice restraints on Env, facilitating its clustering and promoting fusion activity.

biochemistry↗

bioRxiv2026

Structure of HIV-1 Env glycoprotein on virions reveals an alternative fusion subunit organization and native membrane coupling

An effective vaccine for Human Immunodeficiency Virus type-1 (HIV-1) has yet to be developed, and detailed characterization of functional Env glycoprotein, the primary antigenic target on virions, has remained elusive. While engineered Env trimers recapitulate many aspects of functional Env, key differences in antigenicity and dynamic behavior have been reported. Here, cryo-electron tomography and subtomogram averaging of HIV-1 virus-like particles (VLPs) revealed conformational differences in critical membrane-proximal regions compared to soluble Envs. Hydrogen/Deuterium-Exchange Mass Spectrometry and Molecular Dynamics captured dynamic profiles of membrane-bound Env and identified critical interactions with membrane. We show that disruption of the viral membrane results in relaxation of Env to a form that resembles engineered, soluble trimers. Additionally, Env from mature and immature VLPs exhibit only minor conformational differences, while surface clustering on virions changes significantly. These studies provide new insights into the essential role the membrane plays in maintaining Env in its native conformational form.

biochemistry↗

bioRxiv2025

Tau4RD fibril polymorphism is imprinted during early aggregation

Microtubule-associated protein tau forms characteristic fibrillar species in many neurodegenerative diseases. Neurofibrillary tangles, tau deposits observed in Alzheimers disease (AD), contain a mixture of amyloid-type polymorphic fibrils called paired helical filaments (PHFs) and straight filaments. The formation of heterogenous fibril populations is observed in other diseases and when tau aggregation is induced in vitro with polyanionic species. This suggests that taus structural transition from a conformational ensemble to various amyloid morphologies is a controlled and, therefore, controllable process. Despite many years of work toward describing aggregation intermediates that could address open questions such as whether fibril polymorphism is imprinted at the start of aggregation or arises due to conformational conversions, our understanding of amyloid structure remains predominantly based on observations of mature fibrils. It is unclear whether these processes are mutually exclusive and to what extent we can bias intermediate conformations toward less toxic states. Here to address the challenge of studying aggregation intermediates and taus structural conversion, we apply pulsed hydrogen-deuterium exchange with mass spectrometry (pulsed HDX-MS), which revealed differences in the subpopulations formed by tau4RD (a truncated tau construct) within seconds of initiating aggregation with polyphosphate and within hours of heparin-induction. This work begins to address the gap in knowledge regarding whether amyloid polymorphism is directly imprinted during nucleation or results from structural rearrangement during later stages of aggregation.

biophysics↗

bioRxiv2025

Host Switching Mutations in H5N1 Influenza Hemagglutinin Suppress Site-specific Activation Dynamics

Increase in the occurrence of human H5N1 spillover infections resulting from dissemination of highly pathogenic avian influenza (HPAI) virus into bird and mammal populations raises concerns about HPAI adapting to become human transmissible. Studies identified hemagglutinin (HA) acid stability and receptor preference as essential traits that shape host tropism. Mutations that increase HA stability and affinity for -2,6-linked sialic acids have been shown to confer airborne transmissibility in a ferret model, however mechanisms of activation of H5 subtype HA have not been probed and the effect of adaptive mutations on HA function has been largely inferred from static structures. Here, we use hydrogen/deuterium-exchange mass spectrometry to dissect activation dynamics for two ancestral HPAI H5 HA, their matched HA with adaptive mutations, and a contemporary H5 HA. By measuring dynamics, we identify variation in active site flexibility among the HA and demonstrate that adaptive mutations result in suppression of fusion peptide dynamics and stabilization of a key subunit interface involved in activation. The contemporary H5 isolated from a recent human spillover case exhibits a relatively protected fusion peptide and moderately depressed pH of activation compared to the HAs examined in this study. Our studies of activation dynamics in the H5 HAs in conjunction with prior analysis of H1 and H3 HA reveal subtype-specific patterns that correlate with adaptive mutation sites and indicate underlying physical constraints on influenza HA adaptation.

biophysics↗

bioRxiv2025

Interprotomer Crosstalk in Mosaic Viral Glycoprotein Trimers Provides Insight into Polyvalent Immunogen Co-assembly

SARS-CoV-2 variants have demonstrated the ability to evade immune responses, leading to waves of infection throughout the pandemic. In response, bivalent mRNA vaccines, encoding the original Wuhan-Hu-1 and emerging variants, were developed to display both spike antigens. To date, it has not been determined whether co-transfection and co-translation of different SARS-CoV-2 variants results in co-assembly of mosaic heterotrimer antigens and how this may affect trimer stability, dynamics, and antigenicity. Understanding whether such mosaic heterotrimers can form and their implications for antigen structure can provide important information to guide future polyvalent vaccine design where multiple variants of an antigen are co-formulated. To investigate this, we purified mosaic spike assemblies of both genetically close (Omicron BA.2 and XBB) and distant (Omicron BA.2 and Wuhan-Hu-1 G614) strains. We found that the stability and integrity of mosaic spike trimers were maintained without misfolding or aggregation. Glycosylation profiles likewise were preserved relative to the homotrimer counterparts. Hydrogen/deuterium-exchange mass spectrometry and biolayer-interferometry were used to investigate the mosaic spike dynamics and any impact on epitope presentation and receptor binding. The Omicron-XBB heterotrimer, sharing a common fusion subunit sequence, retained protomer-specific dynamics similar to the corresponding homotrimers in antigenically important regions. The Omicron-G614 heterotrimer, co-assembling from protomers of divergent fusion subunit sequences, likewise showed overall similar dynamics and conformations in the receptor-binding subunit compared to the homotrimers. However, the incorporation of the Wuhan-Hu-1 G614 protomer led to a stabilizing effect on the relatively unstable Omicron fusion subunit in the heterotrimer. These findings reveal structural dynamic crosstalk in mosaic trimers, suggesting a potential for enhanced immunogen display and important considerations to be aware of in the use of polyvalent nucleic acid vaccines. Author summaryHere we investigated the possibility of immunogen co-assembly from bivalent nucleic acid vaccines with a focus on probing potential impacts on antigen stability and epitope display. We purified mosaic heterotrimers composed of Omicron BA.2 and Wuhan-Hu-1 G614 protomers as well as those composed of Omicron BA.2 and XBB protomers. Both mosaic heterotrimers maintained their structural integrity, morphology, and N-glycosylation profiles. Using hydrogen/deuterium-exchange mass spectrometry, we demonstrated that both types of heterotrimers preserved strain-specific dynamics in critical antigenic regions, comparable to their homotrimer counterparts. Crosstalk between fusion subunits of Omicron BA.2 and Wuhan-Hu-1 G614 revealed a stabilizing effect of the G614 protomer on the inherently less stable fusion subunit in Omicron. Our findings provide insight into the structural dynamic profiles of co-expressed immunogen display, highlighting important considerations for polyvalent formulations that are being pursued to provide broad protection against highly variable pathogens.

microbiology↗

bioRxiv2025

Collective Microbial Effects Drive Toxin Bioremediation and Enable Rational Design

The metabolic activity of microbial communities is essential for host and environmental health, influencing processes from immune regulation to bioremediation. Given this importance, the rational design of microbiomes with targeted functional properties is an important objective. Designing microbial consortia with targeted functions is challenging due to complex community interactions and environmental heterogeneity. Community-function landscapes address this challenge by statistically inferring impacts of species presence or absence on function. Similar to fitness landscapes, community-function landscapes are shaped by both additive effects and interactions (epistasis) among species that influence function. Here, we apply the community-function landscape approach to design synthetic microbial consortia to degrade the toxic environmental contaminant bisphenol-A (BPA). Using synthetic communities of BPA-degrading isolates, we map community-function landscapes across increasing BPA concentrations, where higher BPA means greater toxicity. As toxicity increases, so does epistasis, indicating that collective effects become more important in degradation. Further, we leverage landscapes to rationally design communities with predictable BPA degradation dynamics in vitro. Remarkably, designed synthetic communities are able to remediate BPA in contaminated soils. Our results demonstrate that toxicity can drive epistatic interactions in community-function landscapes and that these landscapes can guide microbial consortia design for bioremediation.

microbiology↗

bioRxiv2024

Heterogeneous efflux pump expression underpins phenotypic resistance to antimicrobial peptides

Antimicrobial resistance threatens the viability of modern medical interventions. There is a dire need of developing novel approaches to counter resistance mechanisms employed by starved or slow-growing pathogens that are refractory to conventional antimicrobial therapies. Antimicrobial peptides have been advocated as potential therapeutic solutions due to low levels of genetic resistance observed in bacteria against these compounds. However, here we show that subpopulations of stationary phase Escherichia coli and Pseudomonas aeruginosa survive tachyplesin treatment without genetic mutations. These phenotypic variants display enhanced efflux activity to limit intracellular peptide accumulation. Differential regulation of genes involved in outer membrane vesicle section, membrane modification, and protease activity were also found between phenotypically resistant and susceptible cells. We discovered that formation of these phenotypic variants could be prevented by administering tachyplesin in combination with sertraline, a clinically used antidepressant, suggesting a novel approach for combatting antimicrobial-refractory stationary phase bacteria.

microbiology↗

bioRxiv2024

Functional phases encode the response of the soil microbiome to environmental change

The metabolic activity of soil microbiomes plays a central role in carbon and nitrogen cycling. Given the changing climate, it is important to understand how the metabolism of natural communities responds to environmental change. However, the ecological, spatial, and chemical complexity of soils makes understanding the mechanisms governing the response of these communities to perturbations challenging. Here, we overcome this complexity by using dynamic measurements of metabolism in microcosms and modeling to reveal regimes where a few key mechanisms govern the response of soils to environmental change. We sample soils along a natural pH gradient, construct >1500 microcosms to perturb the pH, and quantify the dynamics of respiratory nitrate utilization, a key process in the nitrogen cycle. Despite the complexity of the soil microbiome, a minimal mathematical model with two variables, the quantity of active biomass in the community and the availability of a growth-limiting nutrient, quantifies observed nitrate utilization dynamics across soils and pH perturbations. Across environmental perturbations, changes in these two variables give rise to three functional regimes each with qualitatively distinct dynamics of nitrate utilization over time: a regime where acidic perturbations induce cell death that limits metabolic activity, a nutrientlimiting regime where nitrate uptake is performed by dominant taxa that utilize nutrients released from the soil matrix, and a resurgent growth regime in basic conditions, where excess nutrients enable growth of initially rare taxa. The underlying mechanism of each regime is predicted by our interpretable model and tested via amendment experiments, nutrient measurements, and sequencing. Further, our data suggest that the long-term history of environmental variation in the wild influences the transitions between functional regimes. Therefore, quantitative measurements and a mathematical model reveal the existence of qualitative regimes that capture the mechanisms and dynamics of a community responding to environmental change.

ecology↗

bioRxiv2023

Combinatorial immune refocusing within the influenza hemagglutinin head elicits cross-neutralizing antibody responses

The head domain of influenza hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies during infection and vaccination. Here we evaluated a series of immunogens that combined several immunofocusing techniques for their ability to enhance the functional breadth of vaccine-elicited immune responses. We designed a series of "trihead" nanoparticle immunogens that display native-like closed trimeric heads from the HAs of several H1N1 influenza viruses, including hyperglycosylated variants and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the periphery of the receptor binding site (RBS). Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicited higher HAI and neutralizing activity against vaccine-matched and -mismatched H1 viruses than corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation, indicating that both of these engineering strategies contributed to improved immunogenicity. By contrast, mosaic nanoparticle display and antigen hypervariation did not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Serum competition assays and electron microscopy polyclonal epitope mapping revealed that the trihead immunogens, especially when hyperglycosylated, elicited a high proportion of antibodies targeting the RBS, as well as cross-reactive antibodies targeting a conserved epitope on the side of the head. Our results yield important insights into antibody responses against the HA head and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses. HIGHLIGHTSO_LIGeneralization of trihead antigen platform to several H1 hemagglutinins, including hyperglycosylated and hypervariable variants C_LIO_LITrimer-stabilizing mutations in trihead nanoparticle immunogens lead to lower levels of non-neutralizing antibody responses in both mice and rabbits C_LIO_LIHyperglycosylated triheads elicit higher antibody responses against broadly neutralizing epitopes C_LI

immunology↗

bioRxiv2023

Novel mRNA-based VP8* vaccines against rotavirus are highly immunogenic in rodents

Despite the availability of live-attenuated oral vaccines, rotavirus remains a major cause of severe childhood diarrhea worldwide. Due to the growing demand for parenteral rotavirus vaccines, we developed novel mRNA-based vaccine candidates targeting the viral spike protein VP8*. Our monomeric P2 (universal T cell epitope)-VP8* mRNA design is equivalent to a protein vaccine currently in clinical development, while LS (lumazine synthase)-P2-VP8* was designed to form nanoparticles. Cyro-electron microscopy and western blotting-based data presented here suggest that proteins derived from LS-P2-VP8* mRNA are secreted in vitro and self-assemble into 60-mer nanoparticles displaying VP8*. mRNA encoded VP8* was immunogenic in rodents and introduced both humoral and cellular responses. LS-P2-VP8* induced superior humoral responses to P2-VP8* in guinea pigs, both as monovalent and trivalent vaccines, with encouraging responses detected against the most prevalent P genotypes. Overall, our data provide evidence that trivalent LS-P2-VP8* represents a promising mRNA-based next-generation rotavirus vaccine candidate.

immunology↗

bioRxiv2023

Single-cell RNA sequencing reveals dysregulated fibroblast subclusters in prurigo nodularis

Prurigo nodularis (PN) is an intensely pruritic, chronic inflammatory skin disease that disproportionately affects black patients. However, the pathogenesis of PN is poorly understood. We performed single-cell transcriptomic profiling, ligand receptor analysis and cell trajectory analysis of 28,695 lesional and non-lesional PN skin cells to uncover disease-identifying cell compositions and genetic characteristics. We uncovered a dysregulated role for fibroblasts (FBs) and myofibroblasts as a key pathogenic element in PN, which were significantly increased in PN lesional skin. We defined seven unique subclusters of FBs in PN skin and observed a shift of PN lesional FBs towards a cancer-associated fibroblast (CAF)-like phenotype, with WNT5A+ CAFs increased in the skin of PN patients and similarly so in squamous cell carcinoma (SCC). A multi-center PN cohort study subsequently revealed an increased risk of SCC as well as additional CAF-associated malignancies in PN patients, including breast and colorectal cancers. Systemic fibroproliferative diseases were also upregulated in PN patients, including renal sclerosis and idiopathic pulmonary fibrosis. Ligand receptor analyses demonstrated increased FB1-derived WNT5A and periostin interactions with neuronal receptors MCAM and ITGAV, suggesting a fibroblast-neuronal axis in PN. Type I IFN responses in immune cells and increased angiogenesis/permeability in endothelial cells were also observed. As compared to atopic dermatitis (AD) and psoriasis (PSO) patients, increased mesenchymal dysregulation is unique to PN with an intermediate Th2/Th17 phenotype between atopic dermatitis and psoriasis. These findings identify a pathogenic role for CAFs in PN, including a novel targetable WNT5A+ fibroblast subpopulation and CAF-associated malignancies in PN patients.

bioinformatics↗

bioRxiv2022

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are novel in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1. Significance StatementSARS-CoV-2 infections can result in diverse clinical outcomes, including severe disease. Monoclonal antibodies (mAbs) have been used therapeutically to treat infection, but the emergence of variants has compromised their efficacy. Thus, identifying mAbs that are more durable in the face of SARS-CoV-2 evolution is a pressing need. Here, we describe four new mAbs isolated from a Delta-breakthrough infection, that can potently neutralize diverse variants, including multiple Omicron variants. In addition, one mAb shows broader activity against coronaviruses. The breadth of these mAbs is due to their focus on highly conserved regions of the viral protein antigen, including regions that are required for the virus to enter the cell. These properties make them promising candidates for therapeutic use.

immunology↗

bioRxiv2022

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKVs complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoproteins post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

biophysics↗

bioRxiv2021

Fast bacterial growth reduces antibiotic accumulation and efficacy

Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here we hypothesize that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast growing variants, including efflux that can reduce macrolide accumulation. Using this new knowledge, we phenotypically engineered bacterial populations by eradicating variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.

microbiology↗

bioRxiv2021

Cryo-ET of HIV reveals Env positioning on Gag lattice and structural variation among Env trimers

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1[A] sub-tomogram averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers plus a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

biophysics↗