Biology subjects

Fletcher, M.

Publications and source records attributed to Fletcher, M..

6 recordsLinked to original sources

bioRxiv2024

Chromosomal fusion drives sex chromosome evolution in treehoppers despite long-term X chromosome conservation

Sex chromosomes follow distinct evolutionary trajectories compared to the rest of the genome. In many cases, sex chromosomes (X and Y, or Z and W) significantly differentiate from one another resulting in heteromorphic sex chromosome systems. Such heteromorphic systems are thought to act as an evolutionary trap that prevents subsequent turnover of the sex chromosome system. For old, degenerated sex chromosome systems in which turnover is unlikely, chromosomal fusion with an autosome may be one way that sex chromosomes can refresh their sequence content. We investigated these dynamics using treehoppers (hemipteran insects of the family Membracidae), which ancestrally have XX/X0 sex chromosomes. We assembled the first chromosome-level treehopper genome from Umbonia crassicornis and employed comparative genomic analyses of 12 additional treehopper species to analyze X chromosome variation across different evolutionary timescales. We find that the X chromosome is largely conserved, with one exception being an X-autosome fusion in Calloconophora caliginosa. We also compare the ancestral treehopper X with other X chromosomes in Auchenorrhyncha (the clade containing treehoppers, leafhoppers, spittlebugs, cicadas, and planthoppers), revealing X conservation across more than 300 million years. These findings shed light on chromosomal evolution dynamics in treehoppers and the role of chromosomal rearrangements in sex chromosome evolution. SignificanceThe evolutionary forces underlying sex chromosome stability versus turnover have been challenging to disentangle. We present the first chromosome-level treehopper genome and find evidence of long-term X chromosome conservation within treehoppers - and among treehoppers and other hemipteran insects. A key exception is the evolution of neo-XX/XY sex chromosomes via an X-autosome fusion. Sex chromosome-autosome fusions may play an important role in the evolution of otherwise trapped (i.e., old and degenerated) sex chromosome systems.

evolutionary biology↗

bioRxiv2021

A microfluidic platform for sequential assembly and separation of synthetic cell models

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and by-products, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a stream of vesicles suspension is partitioned into three fractions - purified GUVs, residual components, and a washing solution. Using our purification approach, we showed that giant vesicles can be separated from various residues - that range in size and chemical composition - with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

synthetic biology↗

bioRxiv2021

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, peptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions - their membranes. However, suitable assays to quantify the efficacy of peptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of peptide antibiotics. We reveal strong correlations between each peptides physicochemical activity at the membrane level and biological activity at the cellular level by assaying the membranolytic activities of the antibiotics on hundreds of individual giant lipid vesicles, and quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

microbiology↗

bioRxiv2021

Measuring thousands of single vesicle leakage events reveals the mode of action of antimicrobial peptides

Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, existing characterization assays lack the resolution necessary to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations, and related the observed modes with the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner, and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

biophysics↗

bioRxiv2021

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms. Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

microbiology↗

bioRxiv2018

Global transcriptional regulation of innate immunity in C. elegans

The nematode Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1- ATF-7 signaling reveals global control over the innate immune response to infection through a single transcriptional regulator in a simple animal host.\n\nAuthor SummaryInnate immunity is the first line of defense against invading microbes across metazoans. Caenorhabditis elegans lacks adaptive immunity and is therefore particularly dependent on mounting an innate immune response against pathogens. A major component of this response is the conserved PMK-1/p38 MAPK signaling cascade, the activation of which results in phosphorylation of the bZIP transcription factor ATF-7. Signaling via PMK-1 and ATF-7 causes broad transcriptional changes including the induction of many genes that are predicted to have antimicrobial activity including C-type lectins and lysozymes. In this study, we show that ATF-7 directly regulates the majority of innate immune response genes upon pathogen infection of C. elegans, and demonstrate that many ATF-7 targets function to promote pathogen resistance.

immunology↗