Biology subjects

Allen, R. D.

Publications and source records attributed to Allen, R. D..

8 recordsLinked to original sources

bioRxiv2025

The staphylococcal type VII secretion system delays macrophage cell death through modulating multiple cell death pathways

The major human pathogen Staphylococcus aureus is a facultatively intracellular bacterium that can replicate and survive within a range of host cells including macrophages. While macrophage-mediated immune modulation is pivotal in S. aureus pathogenesis, bacterial manipulation of macrophage pathways remains poorly understood. The specialised type VII secretion system (T7SSb) is an important virulence-associated factor and immune modulator during staphylococcal infection, although, unlike its mycobacterial counterparts, its role in controlling staphylococcal-macrophage interactions remains unclear. Employing high-resolution time-lapse imaging, we demonstrated an induction of cell death corpses or pore-induced cellular traps (PITs) during S. aureus infection of macrophages in vitro, with a higher number of PITs forming in macrophages infected with S. aureus lacking EssC ({Delta}essC), a central T7SS transporter. Interestingly, {Delta}essC-infected macrophages displayed increased bacterial escape compared to wild type (WT)-infected cells, implicating a role for T7SS in delaying macrophage cell death. {Delta}essC-infected macrophages were also efferocytosed less compared to WT in vitro. We investigated the induction of cell death signalling markers in WT and {Delta}essC-infected macrophages and demonstrated that the T7SS delayed the induction of key cell death pathways, necroptosis and pyroptosis. A dual RNAseq analysis of WT- and {Delta}essC-infected macrophages further indicated a T7SS-dependent increase in ferroptosis later in infection, along with modulation of chemokines. We demonstrated that individual T7SS effectors, EsxA and EsxC, which were induced within macrophages, could individually delay macrophage cell death. Furthermore, in a murine skin infection model, we showed that effector and transporter mutants have distinct S. aureus infection outcomes. Our findings suggest for the first time a key role for T7SSb proteins in controlling macrophage cell death, which impacts staphylococcal survival within the host during infection. ImportanceStaphylococcus aureus is a major hospital and community associated pathogen which causes an array of infections. S. aureus can survive within immune cells, which may provide a niche for this pathogen to both persist and disseminate. Here we report a role for the staphylococcal type VII secretion system b, which secretes effectors that have been associated with bacterial virulence, in delaying macrophage cell death induced in response to this pathogen. T7SS functions through interfering with inflammatory cell death pathways, impacting the local cellular and immune environment. Our findings thus highlight a protective role for this system in macrophages, distinct to functions reported for the mycobacterial T7SS, and may implicate similar functions for T7SS proteins from related pathogens. Controlling macrophage cell death could be a critical mechanism by which S. aureus modulates the local immune responses, enabling its survival within the host,

microbiology↗

bioRxiv2022

Elucidating the unknown transcriptional responses and PHR1 mediated biotic and abiotic stress tolerance during phosphorus-limitation

Phosphorus (P) limitation in the majority of world soils is a major constraint for plant growth and crop productivity. RNA sequencing was used to discover novel P-responsive gene transcripts (PRGT) in leaves and roots of Arabidopsis. Hisat StringTie and Cufflinks TopHat transcript assembler were used to analyze reads and identify 1,074 PRGTs with a >5-fold altered abundance during P-limitation. Interestingly, 60% of these transcripts were not previously reported. Among the novel PRGT, 106 were from unannotated genes, and some were among the most P-responsive, including At2g36727 which encodes a novel microRNA. Annotated novel PRGTs encode for transcription factors, microRNAs, small signaling peptides, long non-coding RNAs, defense-related proteins, and transporters, along with proteins involved in many biological processes. We identified several genes that undergo alternative splicing during P-limitation, including a novel miR399 resistant splice variant of PHOSPHATE2 (PHO2.2). Several novel P-responsive genes were regulated by PHOSPHATE STARVATION RESPONSE1 (PHR1), PHR1-LIKE 1 (PHL1) and PHO2. We discovered that P-limited plants show increased resistance to pathogens and drought stress mediated by PHR1-PHL1. Identification of novel P-responsive transcripts and the discovery of the influence of P-limitation on biotic and abiotic stress adds a significant component to our understanding of plant P-signaling. HighlightPhosphorus limitation elicits the expression of several novel genes including many previously unannotated genes, noncoding RNAs, small peptides and alternatively spliced RNAs, and leads to enhanced disease and drought tolerance.

plant biology↗

bioRxiv2021

Protein-based RBD-C-tag COVID-19 Vaccination Candidate Elicits Protection Activity against SARS-COV-2 Variant Infection

The identification of a vaccination candidate against COVID-19 providing protecting activity against emerging SARS-COV-2 variants remains challenging. Here, we report protection activity against a spectrum of SARS-COV-2 and variants by immunization with protein-based recombinant RBD-C-tag administered with aluminum-phosphate adjuvant intramuscularly. Immunization of C57BL/6 mice with RBD-C-tag resulted in the in vivo production of IgG antibodies recognizing the immune-critical spike protein of the SARS-COV-2 virus as well as the SARS-COV-2 variants alpha ("United Kingdom"), beta ("South Africa"), gamma ("Brazil/Japan"), and delta ("India") as well as wt-spike protein. RBD-C-tag immunization led to a desired Th1 polarization of CD4 T cells producing IFN{gamma}. Importantly, RBD-C-tag immunization educated IgG production delivers antibodies that exert neutralizing activity against the highly transmissible SARS-COV-2 virus strains "Washington", "South Africa" (beta), and "India" (delta) as determined by conservative infection protection experiments in vitro. Hence, the protein-based recombinant RBD-C-tag is considered a promising vaccination candidate against COVID-19 and a broad range of emerging SARS-COV-2 virus variants.

microbiology↗

bioRxiv2020

Protective Effects of STI-2020 Antibody Delivered Post-Infection by the Intranasal or Intravenous Route in a Syrian Golden Hamster COVID-19 Model

We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes.

microbiology↗

bioRxiv2020

Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach

SARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a naive phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.

microbiology↗

bioRxiv2020

WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion in Medicago truncatula and Nicotiana sylvestris

Plant specific WOX family transcription factors are known to regulate embryogenesis, meristem maintenance and lateral organ development. Modern clade WOX genes function through a transcriptional repression mechanism, and the intermediate clade transcriptional activator WOX9 functions with the repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. WOX1 homologues STF and LAM1 are required for leaf blade outgrowth in Medicago truncatula and Nicotiana Sylvestris, respectively. Here we show that WOX9 negatively regulates leaf blade outgrowth and functions antagonistically to STF and LAM1. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression of NsWOX9 and knockout by CRISPR/Cas9 genome editing in N. sylvestris resulted in a range of severe leaf blade distortions, indicating that controlled negative regulation by NsWOX9 is required for proper blade development. Our results indicate that direct repression of WOX9 transcriptional activation activity by the transcriptional repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that a balance between transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in higher plants. One sentence summaryWOX9 negatively regulates blade outgrowth antagonizing STF function but directly repressed by STF indicating WOX-mediated homeostasis in cell proliferation and differentiation during leaf morphogenesis.

plant biology↗

bioRxiv2019

HSI2/VAL1 and HSL1/VAL2 function redundantly to regulate seed dormancy by controlling DOG1 expression in Arabidopsis

DELAY OF GERMINATION1 (DOG1) represents a major quantitative locus for the genetic regulation of seed dormancy in Arabidopsis. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination. Here, we report that the conserved B3 DNA binding domains of the transcriptional repressors HIGH-LEVEL EXPRESSION OF SUGAR INDICIBLE GENE2/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE1 (HSI2/VAL1) and HSI2-LIKE1/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE2 (HSL1/VAL2), which play critical roles in the developmental transition from seed maturation to seedling growth, interact with RY elements in the DOG1 proximal promoter leading to repression of DOG1 transcription during germination and seedling establishment. DOG1 expression is partially de-repressed in hsi2/val1 (hsi2-2) but not in hsl1/val2 (hsl1-1) knockout mutants and is strongly upregulated in a hsi2/val1 hsl1/val2 double mutant, indicating that HSI2/VAL1 and HSL1/VAL2 act redundantly to repress DOG1 expression. HSI2/VAL1 and HSL1/VAL2 form homo- and hetero-dimers in vivo, and dimerization is dependent on the HSI2/VAL1 PHD-like domain. Complementation of hsi2-2 with HSI2/VAL1 harboring a disrupted plant homeodomain (PHD)-like domain results in stronger de-repression of DOG1 expression than the hsi2-2 knockout, indicating that the PHD-like domain plays a critical role in mediating functional interactions between HSI2/VAL1 and HSL1/VAL2. Both HSI2/VAL1 and HSL1/VAL2 interact with components of polycomb repressive complex 2 (PRC2), including CURLY LEAF and MULTICOPY SUPPRESSOR OF IRA1 (MSI1), along with LIKE HETERCHROMATIN PROTEIN 1 (LHP1), which are involved in the deposition and expansion of histone H3 lysine 27 trimethylation (H3K27me3) marks in repressive chromatin. Thus, HSI2/VAL1 HSL1/VAL2-dependent recruitment of PRC2 leads to silencing of DOG1 through the deposition of H3K27me3.

molecular biology↗

bioRxiv2017

Functional characterization of the ABF gene family in upland cotton (Gossypium hirsutum L.)

The AREB/ABF bZIP transcription factors play a pivotal role in abscisic acid-dependent abiotic stress-responsive gene expression. Despite the perennial damage and reduced productivity that result from water-deficit and unpredictable early season temperature fluctuations, these critical genes have not been previously examined in upland cotton (Gossypium hirsutum). Here, we report the isolation of the G. hirsutum ABF homologs, characterization of their expression patterns in response to abiotic stress treatments, and examination of their functions through heterologous ectopic expression in Arabidopsis. As expected for an allotetraploid, G. hirsutum ABF homologs are present in the genome as homeologous pairs. These genes are differentially expressed, both among the homologs and within the homeologous pairs, in response to exogenous abscisic acid (ABA) application, dehydration, and chilling temperatures. Furthermore, heterologous ectopic expression of many of the G. hirsutum ABF genes in Arabidopsis conferred increased tolerance to water deficit and osmotic stress, as well as cold tolerance, in a gene specific manner. These results indicate the G. hirsutum ABF homologs are functional in Arabidopsis and, as in other species, are likely to play an essential role in the abiotic stress response.\n\nHighlightThe Gossypium hirsutum ABF homeologs are differentially expressed in response to abiotic stress, and their ectopic expression in Arabidopsis can confer increased water deficit tolerance.

plant biology↗