Biology subjects

Boyd, A.

Publications and source records attributed to Boyd, A..

8 recordsLinked to original sources

bioRxiv2023

Whole Transcriptome and Functional Analyses Identify Novel Genes Involved in Meiosis and Fertility in Drosophila melanogaster

Reproductive success requires the development of viable oocytes and the accurate segregation of chromosomes during meiosis. Failure to segregate chromosomes properly can lead to infertility, miscarriages, or developmental disorders. A variety of factors contribute to accurate chromosome segregation and oocyte development, such as spindle assembly and sister chromatid cohesion. However, many proteins required for meiosis remain unknown. In this study, we aimed to identify and characterize novel meiotic and fertility genes using the genome of Drosophila melanogaster. To accomplish this goal, genes upregulated within meiotically active tissues were identified. About 200 genes with no known function were silenced using RNA interference (RNAi), and the effects on meiosis and fertility were assessed. We identified 65 genes that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, we identified novel genes that are crucial for meiosis and oocyte development but have not been extensively studied in human or model organisms. Understanding the function of these genes will be an important step towards the understanding of their biological significance during reproduction. Author SummaryIn this study, we aimed to identify and characterize novel meiotic and fertility genes within the genome of Drosophila melanogaster. We identified 65 genes that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, we identified novel genes that are crucial for meiosis and oocyte development, making them strong candidates for future studies to characterize their functions.

genetics↗

bioRxiv2022

Olfactory microvillar tuft cells direct neurogenesis during allergic inflammation

The olfactory neuroepithelium serves as a sensory organ for odors and is part of the nasal mucosal barrier. Olfactory sensory neurons are surrounded and supported by epithelial cells. A subset of these, microvillous cells (MVCs), are strategically positioned at the apical surface but their specific functions are still enigmatic and their relationship to the rest of the solitary chemosensory cell family is unclear. Here, we establish that the larger family of MVCs comprises tuft cells and ionocytes in both mice and humans. Olfactory TRPM5+ tuft-MVCs share a core transcriptional profile with the chemosensory tuft family, prominently including the machinery for lipid mediator generation. Integrating analysis of the respiratory and olfactory epithelium, we define the unique receptor expression of TRPM5+ tuft-MVC compared to the G[a]-gustducin+ respiratory tuft cells and characterize a new population of glandular DCLK1+ tuft cells. To establish how allergen sensing by tuft-MVCs might direct olfactory mucosal responses, we employed an integrated single-cell transcriptional and protein analysis. We defined a remodeling olfactory epithelial switch pathway with induction of Chil4 and a distinct pathway of proliferation of the quiescent olfactory horizontal basal stem cell (HBC), both triggered in the absence of significant olfactory apoptosis. While the Chil4 pathway was dependent on STAT6 signaling and innate lymphocytes, neither were required for HBC proliferation. HBC proliferation was dependent on tuft-MVCs, establishing these specialized epithelial cells as both sensors for allergens and regulators of olfactory stem cell responses. Together our data provide high resolution characterization of the nasal tuft cell heterogeneity and uncover a novel mechanism by which TRPM5+ tuft cells direct the olfactory mucosal response to allergens. One Sentence SummaryWe identify the enigmatic TRPM5+ olfactory microvillous cells as tuft cells, and show their functional role as regulators of olfactory stem cell proliferation in response to environmental signals.

immunology↗

bioRxiv2022

In vitro evolution of Listeria monocytogenes reveals selective pressure for loss of SigB and AgrA function at different incubation temperatures.

The alternative sigma factor B ({sigma}B) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the General Stress Response. We previously showed that {sigma}B loss-of-function mutations arise frequently in strains of L. monocytogenes, and suggested that mild stresses might favour the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42{degrees}C) or lower (30{degrees}C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42{degrees}C. However, at 30{degrees}C independent alleles in the agr locus arose, resulting in the inactivation of the Agr quorum sensing. Colonies of both sigB- and agr- strains exhibited a greyer colouration on 7-days-old agar plates compared with the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB- strains outcompeted the parental strain at 42{degrees}C, but not at 30{degrees}C, whilst agr- strains showed a small increase in competitive fitness at 30{degrees}C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of {sigma}B or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. ImportanceTo withstand environmental aggressions L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B ({sigma}B). However, {sigma}B becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to {sigma}B loss-of-function alleles. Temperatures of 42{degrees}C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of {sigma}B loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30{degrees}C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of {sigma}B in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

molecular biology↗

bioRxiv2022

The Vibrio vulnificus stressosome is dispensable in nutrient-replete conditions.

The stressosome is a protein complex that has been demonstrated to sense environmental stresses and mediate the stress response in several Gram-positive bacteria, through the activation of the alternative sigma factor SigB. The in vivo characterisation of this complex has never been performed in Vibrio vulnificus or any other bacteria that do not possess SigB. The elucidation of the role of the stressosome in V. vulnificus would provide elements to elaborate a functional model of the complex in a Gram-negative bacterium and identify the regulatory output in the absence of SigB. The stressosome locus is only found in 44% of Vibrio vulnificus isolates raising the question as to whether the role of stressosome is essential or modulatory in this bacterial species. In this work, the expression of the stressosome genes was proven in nutrient-replete conditions and the co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated. Moreover, the construction of a stressosome mutant lacking the four genes constituting the stressosome complex allowed us to examine the role of this complex in vivo. The initial established mutagenesis strategy relied on rifampicin-resistant V. vulnificus to select recombinant bacteria. Our data clearly showed that the influence of the RifR allele on stress and virulence characteristics overshadowed any effects of the stressosome. Therefore, we established an alternative mutagenesis strategy with a non-modified V. vulnificus parental strain and a DAP auxotrophic E. coli donor strain. Extensive phenotypic characterisation of the successfully-generated mutant in nutrient-replete conditions showed that the stressosome does not significantly contribute to the growth, of V. vulnificus. The stressosome did not modulate the response of V. vulnificus to the range of stresses tested - Ethanol, osmolarity, temperature, and salinity. Furthermore, the stressosome is dispensable for motility and exoenzyme production of V. vulnificus.

microbiology↗

bioRxiv2021

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B ({sigma}B), whose role in protection against acid stress is well established. However, the mechanisms leading to its activation by low pH are unknown. Here, we investigated the involvement of the stressosome, a sensory organelle, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated {sigma}B and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was present but its remaining paralogues were genetically inactivated retained the ability to induce {sigma}B activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1 T175A, T209A, T241A) and RsbS (rsbS S56A), or in the active site of the stressosome kinase RsbT (rsbT N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1 T175A variant, retained a near-wild type phenotype. The rsbR1 T209A and rsbR1 T241A mutants displayed constitutive {sigma}B activity. Mild acid shock upregulates invasion genes and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of {sigma}B in L. monocytogenes. Furthermore, RsbR1 can function independently of its paralogues and that signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle. Author summaryThe stress sensing organelle known as the stressosome, found in many bacterial and archaeal lineages, plays a crucial role in both stress tolerance and virulence in the food-borne pathogen Listeria monocytogenes. However, the mechanisms that lead to its activation and the subsequent activation of the general stress response have remained elusive. In this study, we examined the signal transduction mechanisms that operate in the stressosome in response to acid stress. We found that only one of the five putative sensory proteins present in L. monocytogenes, RsbR1, was required for effective transduction of acid tress signals. We further found that phosphorylation of RsbS and RsbR1, mediated by the RsbT kinase, is essential for signal transduction. Failure to phosphorylate RsbS on Serine 56 completely abolished acid sensing by the stressosome, which prevented the development of adaptive acid tolerance. The acid-induced activation of internalin gene expression was also abolished in mutants with defective stressosome signalling, suggesting a role for the stressosome in the invasion of host cells. Together the data provide new insights into the mechanisms that activate the stressosome in response to acid stress and highlight the role this sensory organelle plays in virulence.

molecular biology↗

bioRxiv2021

Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically drivelung type 2 inflammation

Aeroallergen sensing by airway epithelial cells can trigger pathogenic immune responses leading to chronic type 2 inflammation, the hallmark of airway diseases such as asthma. Airway tuft cells are specialized chemosensory epithelial cells and the dominant source of the epithelial cytokine IL-25 in the trachea and of cysteinyl leukotrienes (CysLTs) in the naive murine nasal mucosa. The interaction of IL-25 and CysLTs and the contribution of tuft cell-derived CysLTs to the development of allergen-triggered inflammation in the airways has not been clarified. Here we show that inhalation of LTC4 in combination with a subthreshold dose of IL-25 leads to dramatic synergistic induction of type 2 inflammation throughout the lungs, causing rapid eosinophilia, dendritic cell (DC) and inflammatory type 2 innate lymphoid cell (ILC2) expansion, and goblet cell metaplasia. While lung eosinophilia is dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells require signaling through both CysLT1R and CysLT2R. Tuft cell-specific deletion of the terminal enzyme requisite for CysLT production, Ltc4s, was sufficient to reduce both the innate immune response in the lung - eosinophilia, KLRG1+ ILC2 activation and DC recruitment - and the systemic immune response in the draining lymph nodes after inhalation of the mold aeroallergen Alternaria. Our findings identify surprisingly potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs. One Sentence SummaryTuft cells produce two highly synergistic mediators: LTC4 and IL-25 to cooperatively induce allergen-driven airway inflammation.

immunology↗

bioRxiv2020

Investigating expression of a human optimized cas9 transgene in Neurospora crassa

The CRISPR-associated Cas9 enzyme is used in molecular biology to engineer the genomes of a wide range of organisms. While Cas9 can be injected or transfected into a target cell to achieve the desired goal, there are situations where stable expression of Cas9 within a target organism is preferable. Here, we show that the model filamentous fungus Neurospora crassa is recalcitrant to heterologous expression of a human-optimized version of Streptococcus pyogenes cas9. Furthermore, partial optimization of cas9 by synonymous codon exchange failed to improve its expression in the fungus. Finally, we show that transgene expression can be detected when cas9Hs sequences are placed in the 3 UTR regions of transgene-derived mRNAs, but not when the same sequences are in the translated part of the transgene-derived mRNA. This finding suggests that the primary obstacle to high cas9Hs expression levels in N. crassa is translational in nature.

molecular biology↗

bioRxiv2020

Alpha globin variation in the long-tailed macaque suggests malaria selection

Human haemoglobin variants, such as sickle, confer protection against death from malaria; consequently, frequencies of such variants are often greatly elevated in humans from malaria endemic regions. Among non-human primates, the long-tailed macaque, Macaca fascicularis, also displays substantial haemoglobin variation. Almost all M. fascicularis haemoglobin variation is in the alpha globin chain, encoded by two linked genes: HBA1 and HBA2. We demonstrate that alpha globin variation in M. fascicularis correlates with the strength of malaria selection. We identify a range of missense mutations in M. fascicularis alpha globin and demonstrate that some of these exhibit a striking HBA1 or HBA2 specificity, a pattern consistent with computational simulations of selection on genes exhibiting copy number variation. We propose that M. fascicularis accumulated amino acid substitutions in its alpha globin genes under malaria selection, in a process that closely mirrors, but does not entirely converge with, human malaria adaptation.

evolutionary biology↗