Biology subjects

Rustandi, R. R.

Publications and source records attributed to Rustandi, R. R..

3 recordsLinked to original sources

bioRxiv2024

Enabling functionality and translation fidelity characterization of mRNA-based vaccines with a platform-based, antibody-free mass spectrometry detection approach

The success of mRNA-based therapeutics and vaccines can be attributed to their rapid development, adaptability to new disease variants, and scalable production. Modified ribonucleotides are often used in mRNA-based vaccines or therapeutics to enhance stability and reduce immunogenicity. However, substituting uridine with N1-methylpseudouridine has recently been shown to result in +1 ribosomal frameshifting that induces cellular immunity to the translated off-target protein. To accelerate vaccine development, it is critical to have analytical methods that can be rapidly brought online to assess the functionality and translation fidelity of mRNA constructs. Here, a platform-based, antibody-free method was developed using cell-free translation (CFT) and liquid chromatography-tandem mass spectrometry (MS) that can detect, characterize, and provide relative quantification of antigen proteins translated from mRNA vaccine drug substance. This workflow enabled the evaluation of mRNA subjected to thermal stress as well as bivalent (i.e., two mRNA encoding different antigen variants) drug substance. Additionally, the MS detection approach exhibited high sensitivity and specificity by accurately identifying all six translated proteins and their relative abundances in a dose-dependent manner following transfection of human cells with a hexavalent mRNA mixture encapsulated in lipid nanoparticles (LNPs), despite significant protein sequence homology. Expanding on these efforts, we show the utility of the CFT-MS approach in identifying the presence and junction of +1 ribosomal frameshifting resulting from N1-methylpseudouridation. Overall, this CFT-MS methodology offers a valuable analytical tool for the development and production of mRNA-based vaccines by facilitating the evaluation of mRNA quality and functionality while ensuring accurate translation of antigen proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/594137v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e4c7a7org.highwire.dtl.DTLVardef@1efddd1org.highwire.dtl.DTLVardef@cbfcbaorg.highwire.dtl.DTLVardef@1ae1acc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Endosomal activation of the Clostridioides difficile binary toxin is Ca2+-dependent

The C. difficile binary toxin (CDT) enters host cells via endosomal delivery like many other AB-type binary toxins. In this study, the cell-binding component of CDT, termed CDTb, was found to bind and form pores in lipid bilayers upon depleting free Ca2+ ion concentrations, and not by lowering pH, as found for other binary toxins (i.e., anthrax). Cryoelectron microscopy, nuclear magnetic resonance spectroscopy, surface plasmon resonance, electrochemical impedance spectroscopy, CDT toxicity studies, and site directed mutagenesis show that dissociation of Ca2+ from a single site in receptor binding domain 1 (RBD1) of CDTb is consistent with a molecular mechanism in which Ca2+ dissociation from RBD1 induces a "trigger" via conformational exchange that enables CDTb to bind and form pores in endosomal membrane bilayers as free Ca2+ concentrations decrease during CDT endosomal delivery. One-Sentence SummaryCa2+ dissociation and unfolding of a receptor binding domain in CDTb activates pore-formation by the C. difficile binary toxin.

biophysics↗

bioRxiv2019

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Targeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 [A]) and an asymmetric form (AsymCDTb; 2.84 [A]). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI. SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

biophysics↗