Biology subjects

Schumacher, M. L.

Publications and source records attributed to Schumacher, M. L..

6 recordsLinked to original sources

bioRxiv2026

Proteolytic feedback of quorum sensing promotes infection by Pseudomonas aeruginosa

Quorum sensing (QS) enables bacteria to coordinate collective behaviors in response to population density. However, the mechanisms by which QS circuits are reset or attenuated remain poorly understood. In Pseudomonas aeruginosa, the LuxR-type transcription factor RhlR controls virulence gene expression at high cell density, requiring both its cognate acyl-homoserine lactone signal and the accessory protein PqsE for full activity. Here, we show that RhlR abundance is actively controlled by ATP-dependent proteolysis through a self-limiting negative feedback loop. We identify an N-terminal degron within RhlR that is protected from protease recognition by PqsE binding and is required for degradation by both the Lon and the ClpP1-ClpP2 protease complex. Removal of this degron stabilizes RhlR, advancing the timing of QS-regulated gene expression, enhancing biofilm formation, imposes a competitive fitness cost in vitro, and reduced virulence in an acute mouse lung infection. Mechanistically, RhlR directly activates transcription of clpP2, encoding a subunit of the protease complex required for its own degradation, establishing a self-limiting negative feedback loop. Disruption of RhlR DNA binding stabilizes the receptor through dual effects on reduced clpP2 expression and increased PqsE availability via the loss of RhlR-dependent repression of pqsE. Together, these findings reveal proteolytic turnover as an underappreciated regulatory layer of QS-activation and establish a mechanism for how receptor turnover may facilitate the transition away from high-cell density states.

microbiology↗

bioRxiv2026

The conserved coordination of acyl-homoserine lactone and PqsE signaling defines the RhlR-dependent quorum-sensing network in Pseudomonas aeruginosa clinical isolates

Quorum sensing (QS) enables Pseudomonas aeruginosa to coordinate virulence and biofilm formation through cell density-dependent signaling. In clinical isolates from patients with cystic fibrosis (pwCF), mutations in canonical QS systems such as lasR and rhlI often lead to altered signaling hierarchies that complicate our understanding of QS regulation during chronic infection. Here, we dissect the relative contributions of the autoinducer N-butyryl-L-homoserine lactone (C4HSL) and the protein binding partner PqsE to RhlR-dependent transcription in CF clinical isolates. Using site-directed mutagenesis to generate RhlR and PqsE variants incapable of responding to C4HSL (RhlR A44M) or dimerizing to interact with RhlR (PqsENI), we show that both inputs are essential for the full expression of QS-regulated virulence factors, including pyocyanin and rhamnolipids. Transcriptomic analyses revealed that C4HSL and PqsE co-regulate a conserved set of 28 RhlR-dependent genes, encompassing canonical virulence loci as well as uncharacterized genes that are likely important for adaptation to the CF airway environment. These findings establish that clinical isolates maintain functional QS circuitry reliant on dual activation of RhlR by both C4HSL and PqsE, revealing a conserved regulatory module that underpins pathogenic behavior across genetically diverse isolates. AUTHOR SUMMARYUnderstanding quorum-sensing regulation in clinical isolates of Pseudomonas aeruginosa is essential to determine how the pathogen persists and adapts within the cystic fibrosis lung. While most studies have focused on laboratory strains, chronic isolates exhibit distinct genetic and regulatory adaptations that complicate our ability to generalize quorum sensing function. Our work defines the coordinated roles of C4HSL and PqsE in activating RhlR-dependent gene expression and virulence factor production in isolates from patients with cystic fibrosis. We identify a conserved core of quorum-sensing-regulated genes that remain dependent on both signals despite extensive genomic divergence. These findings highlight that, even within the evolutionary landscape of chronic infection, quorum-sensing signaling through RhlR remains a central and conserved determinant of virulence. By resolving the dual contributions of acyl-homoserine lactone and PqsE-mediated activation, this work provides a mechanistic foundation for future efforts to therapeutically target quorum-sensing pathways in clinical P. aeruginosa infections.

microbiology↗

bioRxiv2026

PhoPQ is an upstream regulator of quorum sensing in small colony variant subpopulations of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that is hospital-endemic, forming biofilms on medical equipment and causing thousands of hospital-acquired infections each year. The success of P. aeruginosa as an opportunistic pathogen is linked to its phenotypic and genotypic adaptability. Relatedly, P. aeruginosa forms a small colony subpopulation in response to stresses like oxygen limitation and antibiotic exposure. Additionally, P. aeruginosa coordinates population-level decisions using a mechanism of cell-cell communication called quorum sensing. P. aeruginosa uses these signaling pathways to control virulence factor production and biofilm formation in the host. We show that certain quorum-sensing mutations promote phenotypic variation; specifically, deletion of lasR and autoinducer modulating mutations in rhlI enhanced small colony formation in a time course-dependent manner. Using transcriptome analyses of isogenic small and large colony variants, we show that small colony formation is driven in part by the PhoPQ two-component signal transduction system in quorum-sensing mutant backgrounds. Specifically, our data show that unphosphorylated PhoP represses rhlR gene expression, and that subsequent de-repression of quorum sensing contributes to the production of virulence factors and the small colony phenotype. In total, these findings provide insight on how mutations evolved by clinical strains might serve as a bet-hedging strategy to promote the formation of a small colony phenotype and alter quorum-sensing signaling within a subpopulation of a community.

microbiology↗

bioRxiv2026

AncientMetagenomeDir dating metadataset highlights need for standardised radiocarbon reporting in ancient DNA

Ancient DNA is a valuable data source for the understanding of our past. However, to effectively interpret this data, it is essential to know the age of the samples from which the DNA is obtained. Although the field of palaeogenomics has been recognised for its robust open data sharing practices, dating information associated with analysed samples is not reported consistently across palaeogenomic studies, nor is it included as metadata in most genetic data repositories. Here, we describe the addition of standardised precise dating information for ancient microbial genomes into the AncientMetagenomeDir metadata repository of published ancient metagenomic samples. This extension currently includes dating information for over 700 ancient microbial genomic datasets, of which 333 are dated using historical, contextual, or stratigraphic methods, and 405 are radiocarbon dated. We quantitatively assess the quality of radiocarbon date reporting and find that, despite established reporting conventions, radiocarbon dating information is often reported inconsistently across ancient metagenomic studies. This new resource provides ancient microbial researchers with standardised dating information that facilitates more accurate and consistent analysis of metagenomic sequencing data. The dataset also highlights the need for greater standardisation of radiocarbon date reporting in original publications in order to allow effective reuse of this and future ancient microbial data.

bioinformatics↗

bioRxiv2025

RhlR quorum-sensing receptor ligand sensitivity regulates the differential expression of phenazine genes in Pseudomonas aeruginosa

Bacteria control individualistic and group behaviors using a form of cell-cell communication called quorum sensing. Quorum sensing relies on the production of chemical signals called autoinducers and the subsequent detection of those signals by a cognate receptor. Many Gram-negative bacteria use the LuxR-type family of transcription factor receptors that bind to acyl-homoserine lactone autoinducer signals to regulate their function as DNA-binding proteins. A subclass of this family of transcription factor receptors requires their cognate autoinducer to fold and dimerize to bind DNA to regulate gene expression and, thus, traits associated with quorum sensing, such as biofilm formation and virulence factor production. Here, we use a chemical-genetic approach to determine the structural basis for ligand selection by the quorum-sensing receptor RhlR from Pseudomonas aeruginosa. The native ligand for RhlR is N-butyryl-L-homoserine lactone, and this protein-ligand interaction is important for initiating gene expression in P. aeruginosa. We determine key residues that drive ligand specificity and selectivity of RhlR to define the role of ligand-driven RhlR-dependent gene regulation of quorum-sensing traits, namely the differential expression of the phenazine genes, which encode the enzymes responsible for the synthesis of the redox-sensitive virulence factor pyocyanin, among other phenazines. Furthermore, we provide a chemical-genetic framework for future studies aimed at disrupting the RhlR-ligand interaction to suppress virulence in P. aeruginosa, an important nosocomial pathogen with widespread antimicrobial resistance.

microbiology↗

bioRxiv2021

Downregulation of Neurodevelopmental Gene Expression in iPSC-Derived Cerebral Organoids Upon Infection by Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a beta herpesvirus that, upon congenital infection, can cause severe birth defects including vision and hearing loss, microcephaly, and seizures. Currently, no approved treatment options exist for in utero infections. We previously demonstrated that HCMV infection decreases calcium signaling responses and alters neuronal differentiation in induced pluripotent stem cell (iPSC) derived neural progenitor cells (NPCs). Here we aimed to determine the impact of infection on the transcriptome in developing human neurons using iPSC-derived 3-dimensional cerebral organoids. We infected iPSC-derived cerebral organoids with HCMV encoding eGFP and sorted cell populations based on GFP signal strength. Significant transcriptional downregulation was observed including in key neurodevelopmental gene pathways in both the GFP (+) and intermediate groups. Interestingly, the GFP (-) group also showed downregulation of the same targets indicating a mismatch between GFP expression and viral infection. Using a modified HCMV virus destabilizing IE 1 and 2 proteins, we still observed significant downregulation of neurodevelopmental gene expression in infected neural progenitor cells. Together, these data indicate that IE viral proteins are not the main drivers of neurodevelopmental gene dysregulation in HCMV infected neural tissues suggesting therapeutically targeting IE gene expression is insufficient to restore neural differentiation and function.

cell biology↗