Biology subjects

Parvate, A.

Publications and source records attributed to Parvate, A..

5 recordsLinked to original sources

bioRxiv2024

Illuminating microbial mat assembly: Cyanobacteria and Chloroflexota cooperate to structure light-responsive biofilms

Microbial mats are stratified communities often dominated by unicellular and filamentous phototrophs within an exopolymer matrix. It is challenging to quantify the dynamic responses of community members in situ as they experience steep gradients and rapid fluctuations of light. To address this, we developed a binary consortium using two representative isolates from hot spring mats: the unicellular oxygenic phototrophic cyanobacterium Synechococcus OS-B (Syn OS-B) and the filamentous anoxygenic phototroph Chloroflexus MS-CIW-1 (Chfl MS-1). We quantified the motility of individual cells and entire colonies and demonstrated that Chfl MS-1 formed bundles of filaments that moved in all directions with no directional bias to light. Syn OS- B was slightly less motile but exhibited positive phototaxis. This binary consortium displayed cooperative behavior by moving further than either species alone and formed ordered arrays where both species aligned with the light source. No cooperative motility was observed when a non-motile pilB mutant of Syn OS-B was used instead of Syn OS-B. The binary consortium also produced more adherent biofilm than individual species, consistent with the close interspecies association revealed by electron microscopy. We propose that cyanobacteria and Chloroflexota cooperate in forming natural microbial mats, by colonizing new niches and building robust biofilms. SignificanceMicrobial mats are dense, layered communities with ancient origins and widespread occurrence, but how they assemble is not well understood. To investigate how microbial motility, physical interactions, and responses to light affect mat assembly, we developed a binary consortium from representative hot spring mat isolates. Individually, the Cyanobacteria and Chloroflexota isolates displayed significant differences in motility and biofilm formation. When combined, the consortium exhibited enhanced motility towards light and formed more robust biofilms. This model consortium approach complements in situ studies by directly testing the role of motility and physical cooperation in shaping microbial mats, and could inform biofilm applications in industrial settings.

microbiology↗

bioRxiv2024

A broadly-neutralizing antibody against Ebolavirus glycoprotein that potentiates the breadth and neutralization potency of other antibodies

Ebolavirus disease (EVD) is caused by multiple species of Ebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Zaire ebolavirus (EBOV). Therefore, mAbs targeting multiple Ebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 [A] cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different Ebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

immunology↗

bioRxiv2024

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Polysaccharides are a major class of natural polymers found abundantly across all major life forms and play a critical role as structural, metabolic, or functional components in biomolecular processes. Some polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs). Despite the fact that the GT-2 family has the maximum number of deposited sequences, the biochemistry of GT-2 family enzymes is still poorly understood due to difficulties associated with GT membrane protein expression, purification, and reconstitution in lipid carriers. Here, we chose Populus tremula x tremuloides cellulose synthase 8 (PttCesA8) and Streptococcus equisimilis hyaluronan synthase (SeHas) as putative family-2-GTs to be expressed in a wheat-germ-based cell-free expression (CFE) system as proteoliposomes. The cell-free products were obtained as reconstituted liposomes directly from CFE reactions at high yields and short processing times compared to other approaches. GT enzymes expression was confirmed using SDS-PAGE and immunoblotting, and the integration of GTs in lipid layers was observed using cryogenic electron microscopy. Both GTs tested were catalytically active when incubated with their respective substrates and cofactors. The Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 {micro}M, and SeHas was 321.51 {micro}M (toward UDP N-Acetyl Glucosamine) and 207.88 {micro}M (toward UDP Glucuronic Acid), respectively. UDP was found to actively inhibit both these GTs with apparent inhibition constants of 10.08 {micro}M and 24.38 {micro}M. Mutation of specific conserved residues in structure-deficit SeHas confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, wheat-germ-based CFE can be used to express functionally active and liposome-reconstituted family-2 GTs at high yields with relative ease to enable classical enzymology assays and will also enable more detailed structural studies in the near future.

bioengineering↗

bioRxiv2022

Structure of the Inmazeb cocktail and resistance to escape against Ebola virus

Monoclonal antibodies can provide important pre- or post-exposure protection against disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. A key concern in use of monotherapy monoclonal antibody products lies in the high risk of mutagenic escape. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, demonstrated efficacy in lessening disease course and improving survival in a randomized, controlled trial. Here we present the cryoEM structure at 3.1 [A] of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with eight Fab fragments of antibodies in the Inmazeb cocktail. This structure allows modeling of previously disordered portions of the glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities, as well as residues that are critical for resistance to escape by each component of this cocktail and other clinically relevant antibodies. We also provide direct evidence that, unlike monotherapy treatments, including those targeting conserved epitopes, the Inmazeb protects against the rapid emergence of EBOV escape mutants and supports the benefit of the combination approach.

biophysics↗

bioRxiv2022

Cryo-EM structure of the diapause chaperone artemin

The protein artemin constitutes over 10% of all protein in Artemia cysts during diapause and acts as both an RNA and protein chaperone. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04 [A] resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.

biochemistry↗