Biology subjects

Garrett, E. M.

Publications and source records attributed to Garrett, E. M..

5 recordsLinked to original sources

bioRxiv2025

Phase variation of Clostridioides difficile colony morphology occurs via modulation of cell division

Phase variation of C. difficile colony morphology occurs via modulation of transcription cmrRST, which encodes a three-protein signal transduction system. Response regulators CmrR and CmrT promote rough colony development, cell elongation and chaining, surface motility, and disease in the hamster model of infection, while impairing swimming motility and biofilm formation. Using RNA-Seq, we identified the CmrR and CmrT-dependent transcriptional differences in rough and smooth colonies. Further analysis showed that CmrT, but not CmrR, is required for differential expression of most of the genes. Two CmrT-regulated genes, herein named mrpA and mrpB, were together sufficient for restoring all CmrT-dependent in vitro phenotypes in a cmrT mutant and alleviating selection of cmr phase ON cells during growth on an agar surface. MrpA and MrpB are uncharacterized proteins with no known function but are highly conserved in C. difficile. Using immunoprecipitation and mass spectrometry to identify interacting partners, we found that MrpA interacts with the septum-site determining protein MinD and several other proteins involved in cell division and cell shape determination. Ectopic expression of mrpAB resulted in atypical cell division, consistent with MrpAB interference with MinD function. Our findings reveal a potential mechanism by which phase variation of CmrRST modulates colony morphology and motility: in cmr phase ON cells, CmrT-mediated expression of mrpAB interferes with normal cell division resulting elongated cells that enable expansion of the population across a surface while limiting swimming motility. AUTHOR SUMMARYC. difficile can reversibly switch between two colony morphology variants that differ in multiple additional phenotypes. Previous work determined that the phenotypic switch occurs through phase variation of the CmrRST signal transduction system, however the CmrRST-regulated genes that mediate these phenotypes were unknown. Here, we identified the CmrR- and CmrT-regulated genes that are differentially expressed between rough and smooth colonies and identified two genes, mrpA and mrpB, that together are sufficient to confer phenotypes associated with cmrRST expression. Protein interaction studies revealed that MrpA interacts with MinD, a protein that helps ensure symmetric cell division in bacteria. Based on these findings, we propose that MrpA disrupts MinD function leading to aberrant cell division, resulting in elongated cells that form rough colonies. This study reveals previously uncharacterized proteins that affect C. difficile cell division and broadly impact the physiology of this pathogen.

microbiology↗

bioRxiv2021

Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile

The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or pdcB switch, determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes. These results indicate that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

microbiology↗

bioRxiv2021

Multiple regulatory mechanisms control the production of CmrRST, an atypical signal transduction system in Clostridioides difficile

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation by site-specific recombination. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the cmr switch, and expression of cmrRST is also regulated by c-di-GMP through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independent of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system. IMPORTANCEClostridioides difficile is a leading cause of hospital-acquired intestinal infections in the U.S. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here we define the complex, multi-level regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.

microbiology↗

bioRxiv2020

Genome-scale metabolic reconstruction analysis of Clostridioides difficile identifies conserved patterns of virulence-related metabolic reprogramming

The pathogen Clostridioides difficile causes toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the US. Due to growing antibiotic resistance and recurrent infection, targeting C. difficile metabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructed C. difficile GENREs for a hyper-virulent isolate (str. R20291) and a historic strain (str. 630), validating both with in vitro and in vivo datasets. Growth simulations revealed significant correlations with measured carbon source usage (PPV [≥] 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated from in vitro and infection conditions, we discovered reliance on the Pentose Phosphate Pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in bacterial pathogenesis. ImportanceClostridioides difficile is a Gram-positive, sporulating anaerobe that has become the leading cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors. In the past, genome-scale metabolic network reconstruction (GENRE) analysis of bacteria has enabled systematic investigation of the genetic and metabolic properties that contribute to downstream virulence phenotypes. With this in mind, we generated and extensively curated C. difficile GENREs for both a well-studied laboratory strain (str. 630) as well as a more recently characterized hyper-virulent isolate (str. R20291). In silico validation of both GENREs revealed high degrees of agreement with experimental gene essentiality and carbon source utilization datasets. Subsequent exploration of context-specific metabolism during both in vitro growth and infection revealed consistent patterns of metabolism which corresponded with experimentally measured increases in virulence factor expression. Our results support that differential C. difficile virulence is associated with distinct metabolic programs related use of carbon sources and provides a platform for identification of novel therapeutic targets.

bioengineering↗

bioRxiv2018

Epigenomic landscape of the human pathogen Clostridium difficile

Clostridioides difficile is a leading cause of health care-associated infections. Although significant progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we performed the first comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observed great epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity whose corresponding gene is highly conserved across our dataset and in all ~300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacted sporulation, a key step in C. difficile disease transmission, consistently supported by multi-omics data, genetic experiments, and a mouse colonization model. Further experimental and transcriptomic analysis also suggested that epigenetic regulation is associated with cell length, biofilm formation, and host colonization. These findings open up a new epigenetic dimension to characterize medically relevant biological processes in this critical pathogen. This work also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomics studies.

genomics↗