Biology subjects

Daniel-Ivad, M.

Publications and source records attributed to Daniel-Ivad, M..

4 recordsLinked to original sources

bioRxiv2026

Structure of Bacillus cereus ZmaM, a peptidase-ABC transporter that secretes and activates zwittermicin A

Bacteria engage in microbial warfare by secreting toxic molecules. These toxins can serve as therapeutics or drug discovery starting points, motivating investigations of their production. Bacteria that produce these toxins must protect themselves, and a common strategy is to synthesize an inactive prodrug that is activated upon secretion. Some prodrugs are capped with N-acyl-[x1D05]-asparagine motifs, which are removed by two types of prodrug peptidases. Type I peptidases are membrane-bound S12 hydrolases; type II peptidases have an additional C-terminal half-ABC transporter module. Recent structures of a type I peptidase revealed how its transmembrane domain completes the substrate-binding site, but little is known about type II peptidases. Here, we show that the B. cereus type II peptidase ZmaM is responsible for both the export of prezwittermicin and its activation into zwittermicin. The structure of homodimeric ZmaM reveals a central type IV ABC transporter flanked by peptidase modules. The periplasmic hydrolase domains interact over the transporter's open outer vestibule. Mutations to intermodule interfaces impact both peptidase and ATP hydrolysis activities, indicating the peptidase and transporter modules are interdependent. Together, our findings reveal an evolutionary strategy to coordinate toxin activation with its export and inform future efforts to harness these systems in biotechnology.

microbiology↗

bioRxiv2024

A cell-free workflow for detecting and characterizing RiPP recognition element-precursor peptide interactions

Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins and peptides. Current methods for studying and engineering PTM installing proteins often suffer from low-throughput experimental techniques. Here we describe a generalizable, in vitro workflow coupling cell-free protein synthesis (CFPS) with AlphaLISA for the rapid expression and testing of PTM installing proteins. We apply our workflow to two representative classes of peptide and protein therapeutics: ribosomally synthesized and post-translationally modified peptides (RiPPs) and conjugate vaccines. First, we demonstrate how our workflow can be used to characterize the binding activity of RiPP recognition elements, an important first step in RiPP biosynthesis, and be integrated into a biodiscovery pipeline for computationally predicted RiPP products. Then, we adapt our workflow to study and engineer oligosaccharyltransferases (OSTs) involved in conjugate vaccine production, enabling the identification of mutant OSTs and sites within a carrier protein that enable high efficiency production of conjugate vaccines. In total, we expect that our workflow will accelerate design-build-test cycles for engineering PTMs.

synthetic biology↗

bioRxiv2023

Identification and targeting of microbial putrescine acetylation in bloodstream infections

The growth of antimicrobial resistance (AMR) has highlighted an urgent need to identify bacterial pathogenic functions that may be targets for clinical intervention. Although severe bacterial infections profoundly alter host metabolism, prior studies have largely ignored alterations in microbial metabolism in this context. Performing metabolomics on patient and mouse plasma samples, we identify elevated levels of bacterially-derived N- acetylputrescine during gram-negative bloodstream infections (BSI), with higher levels associated with worse clinical outcomes. We discover that SpeG is the bacterial enzyme responsible for acetylating putrescine and show that blocking its activity reduces bacterial proliferation and slows pathogenesis. Reduction of SpeG activity enhances bacterial membrane permeability and results in increased intracellular accumulation of antibiotics, allowing us to overcome AMR of clinical isolates both in culture and in vivo. This study highlights how studying pathogen metabolism in the natural context of infection can reveal new therapeutic strategies for addressing challenging infections.

microbiology↗

bioRxiv2017

cAMP-Independent Control Of Twitching Motility In Pseudomonas aeruginosa

FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through activation of the adenylate cyclase, CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimVs cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, required both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds - lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH, or expressing constitutively-active CyaB - pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV-dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimVs role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.\n\nIMPORTANCEPseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility, and are involved in surface sensing which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements - including the PilSR two-component system - to cell poles for optimal function.

microbiology↗