Biology subjects

Jasti, N.

Publications and source records attributed to Jasti, N..

5 recordsLinked to original sources

bioRxiv2026

Microbial mechanistic requirements for eliciting a topical and intranasal immune response

The skin colonist Staphylococcus epidermidis elicits a potent antibody response that can be redirected against an antigen of interest, but this process relies on genetic engineering.1 Here, by adapting bioorthogonal chemistry methods to conjugate antigens to the cell surface, we make the process of generating a commensal vaccine rapid and efficient. A wide variety of bacteria displaying tetanus toxin fragment C (TTFC) elicit an antibody response when applied to mice topically, indicating that the inductive process is not limited to colonists. Colonization occurs at two different sites, the skin and nostril; by colonizing each with S. epidermidis-TTFC, we show that skin colonization yields a moderate IgG response, while nostril colonization elicits a highly potent systemic IgG response and an exuberant IgA response in the nostrils, lungs, and intestine. Two lines of evidence are consistent with the nasal-associated lymphoid tissue (NALT) as the inductive site for nostril colonization: imaging suggesting robust bacterial translocation, and the induction of commensal-specific B cells following colonization. On the skin, TTFC must be conjugated to live S. epidermidis to elicit an antibody response; in the nostrils, live S. epidermidis-TTFC and S. epidermidis mixed with TTFC are equally potent. Commensal vaccination yields a robust response in pet shop mice, and chemical conjugation facilitates antibody responses to a broad array of antigens, including a whole viral capsid and a rotavirus immunogen. Collectively, these findings provide compelling evidence of a translational path for commensal vaccines.

microbiology↗

bioRxiv2026

β-strand complementation within tip initiation complexes licenses assembly of diverse type IV filaments

PilC/PilY1 proteins are tip-located adhesins of type IV pili (T4P) that are critical for T4P function in diverse behaviors including twitching motility, DNA uptake, and host cell adhesion. PilC and PilY1 adhesins are proposed to interact with initiation complexes composed of minor pilins (PilIJK family proteins) to aid in initiation of T4P polymerization, but it has been unclear how PilC/PilY1 proteins promote fiber assembly. We combined structural modeling, genetic, and biochemical experiments using Neisseria gonorrhoeae and Acinetobacter baylyi to delineate how PilC/PilY1 control T4P assembly: a short peptide at the C-terminus of PilC/PilY1 initiates T4P assembly via {beta}-strand complementation with PilK-family minor pilins. This {beta}-strand is necessary and partially sufficient to trigger fiber assembly. In a working model, the PilK-PilC/PilY1 complex is recognized by a preformed PilI-PilJ heterodimer to form a quaternary "licensing complex" that then templates and initiates fiber assembly. In type II secretion systems (T2SS) lacking PilC/PilY1, PilK homologs directly incorporate the terminal {beta}-strand provided by PilC/PilY1 in T4P. Moreover, phylogenetically distinct Tad T4P lack a canonical PilK homolog and instead contain a structurally similar minor pilin-like protein called TadG/CpaL that is important for fiber assembly. We show that CpaL of Caulobacter crescentus Tad T4P acts similarly to the T2SS PilK homolog to provide the C-terminal {beta}-strand required for assembly. Our results explain how PilC/PilY1 can be retained on the fiber tip under enormous tensile loads generated during mechanical shear and T4P retraction and demonstrate how diverse T4P systems employ {beta}-strand complementation to license fiber assembly. SIGNIFICANCEProkaryotic type IV filaments are ancient, diverse, and broadly distributed nanomachines that assemble and retract to execute diverse microbial functions. They include type IV pili and type II secretion systems, mediating toxin secretion, motility, surface adhesion, biofilm formation, DNA uptake, and other functions. Here, we show that two widely conserved subunits of the tip, PilI and PilJ, form a module that recognizes the folding of a {beta}-sheet in a third subunit, PilK. The final {beta}-strand in this sheet can be supplied in trans by the last [~]10 aminoacyl residues of large PilC/PilY1 adhesins, or in cis by PilK itself. In a working model, this recognition results in formation of a PilIJK trimer, which then licenses fiber polymerization through a templating mechanism.

microbiology↗

bioRxiv2025

From sequence to scaffold: computational design of protein nanoparticle vaccines from AlphaFold2-predicted building blocks

Self-assembling protein nanoparticles are being increasingly utilized in the design of next-generation vaccines due to their ability to induce antibody responses of superior magnitude, breadth, and durability. Computational protein design offers a route to novel nanoparticle scaffolds with structural and biochemical features tailored to specific vaccine applications. Although strategies for designing new self-assembling proteins have been established, the recent development of powerful machine learning-based tools for protein structure prediction and design provides an opportunity to overcome several of their limitations. Here, we leveraged these tools to develop a generalizable method for designing novel self-assembling proteins starting from AlphaFold2 predictions of oligomeric protein building blocks. We used the method to generate six new 60-subunit protein nanoparticles with icosahedral symmetry, and single-particle cryo-electron microscopy reconstructions of three of them revealed that they were designed with atomic-level accuracy. To transform one of these nanoparticles into a functional immunogen, we reoriented its termini through circular permutation, added a genetically encoded oligomannose-type glycan, and displayed a stabilized trimeric variant of the influenza hemagglutinin receptor binding domain through a rigid de novo linker. The resultant immunogen elicited potent receptor-blocking and neutralizing antibody responses in mice. Our results demonstrate the practical utility of machine learning-based protein modeling tools in the design of nanoparticle vaccines. More broadly, by eliminating the requirement for experimentally determined structures of protein building blocks, our method dramatically expands the number of starting points available for designing new self-assembling proteins. Significance StatementSelf-assembling protein nanoparticle vaccines have steadily gained traction in both academic and industry vaccine development over the last decade. Recent work has shown that computationally designing new self-assembling proteins allows the structural and functional features of nanoparticle vaccines to be precisely tailored, and that this can significantly affect the vaccine-elicited immune response. To date, designing such nanoparticle vaccines has required the use of known crystal structures as starting points. Here we show how new machine learning tools can be leveraged to design new self-assembling protein nanoparticle immunogens in the absence of experimentally determined structures of the building blocks that elicit strong immune responses in mice.

biochemistry↗

bioRxiv2024

Deep mutational scanning of rabies glycoprotein defines mutational constraint and antibody-escape mutations

Rabies virus causes nearly 60,000 human deaths annually. Antibodies that target the rabies glycoprotein (G) are being developed as post-exposure prophylactics, but mutations in G can render such antibodies ineffective. Here, we use pseudovirus deep mutational scanning to measure how all single amino-acid mutations to G affect cell entry and neutralization by a panel of antibodies. These measurements identify sites critical for rabies Gs function, and define constrained regions that are attractive epitopes for clinical antibodies, including at the apex and base of the protein. We provide complete maps of escape mutations for eight monoclonal antibodies, including some in clinical use or development. Escape mutations for most antibodies are present in some natural rabies strains. Overall, this work provides comprehensive information on the functional and antigenic effects of G mutations that can help inform development of stabilized vaccine antigens and antibodies that are resilient to rabies genetic variation.

microbiology↗

bioRxiv2022

Stac1 Regulates Sensory Stimulus Induced Escape Locomotion

The stac family of genes are expressed by several cell types including neurons and muscles in a wide variety of animals. In vertebrates, stac3 encodes an adaptor protein specifically expressed by skeletal muscle that regulates L-type calcium channels (CaChs) and excitation-contraction coupling. The function of Stac proteins expressed by neurons in the vertebrate CNS, however, is unclear. To better understand neuronal Stac proteins, we identified the stac1 gene in zebrafish. stac1 is expressed selectively in the embryonic CNS including in Kolmer-Agduhr (KA) neurons, the cerebral fluid-contacting neurons (CSF-cNs) in the spinal cord. Previously CSF-cNs in the spinal cord were implicated in locomotion by zebrafish larvae. Thus, expression of stac1 by CSF-cNs and the regulation of CaChs by Stac3 suggest the hypothesis that Stac1 may be important for normal locomotion by zebrafish embryos. We tested to see if optogenetic activation of CSF-cNs was sufficient to induced swimming in embryos as it is in larvae. Indeed, optogenetic activation of CSF-cNs in embryos induced swimming in embryos. Next, we generated stac1-/- null embryos and found that both mechanosensory and noxious stimulus-induced swimming were decreased. We further found that zebrafish embryos respond more vigorously to tactile stimulation in the light compared to the dark. Interestingly, light enhancement of touch-induced swimming was eliminated in stac1 mutants. Thus, Stac1 regulates escape locomotion in zebrafish embryos perhaps by regulating the activity of CSF-cNs. SIGNIFICANCE STATEMENTThe stac genes are a small family of genes found in neurons and muscle in both vertebrates and invertebrates. Stac3 is a muscle protein that controls excitation-contraction coupling via regulation of L-type calcium channels and in humans a STAC3 mutation is responsible for a congenital myopathy. The function of neural Stac proteins, however, is unknown in vertebrates. The findings of this report show that neural stac1 is expressed by cerebral fluid-contacting neurons (CSF-cNs) in the spinal cord of zebrafish embryos and that it is necessary for normal sensory stimulus induced escape swimming. To our knowledge this is the first demonstration of a function for stac genes in neurons in the vertebrate nervous system.

neuroscience↗