Biology subjects

Beckstette, M.

Publications and source records attributed to Beckstette, M..

7 recordsLinked to original sources

bioRxiv2024

RNase-mediated reprogramming of Yersinia virulence

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we report that of 11 tested potential RNases of the intestinal pathogen Yersinia pseudotuberculosis, two, the endoribonuclease RNase III and the exoribonuclease PNPase, repress the synthesis of the master virulence regulator LcrF. LcrF activates the expression of virulence plasmid genes encoding the type III secretion system (Ysc-T3SS) and its substrates (Yop proteins), that are employed to inhibit immune cell functions during infection. Loss of both RNases led to an increase in lcrF mRNA levels and stability. Our work indicates that PNPase exerts its influence via YopD, known to accelerate lcrF mRNA degradation. Loss of RNase III results in the downregulation of the CsrB and CsrC RNAs, leading to increased availability of active CsrA, which has previously been shown to enhance lcrF mRNA translation and stability. Other factors that influence the translation process and were found to be differentially expressed in the RNase III-deficient mutant could support this process. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal towards virulence plasmid-encoded genes. A similar extensive transcriptional reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. This illustrates that RNase III enables immediate coordination of virulence traits, such as Ysc-T3SS/Yops, with other functions required for host-pathogen interactions and survival in the host. Author SummaryBacterial pathogens need to quickly adapt the expression of virulence- and fitness-relevant traits in response to host defenses. Pathogenic Yersinia species rapidly upregulate a type III secretion system (T3SS) to inject antiphagocytic and cell toxic effector proteins, named Yersinia outer proteins (Yops), into attacking immune cells. For this purpose, they display complex and resilient regulatory mechanisms. At the post-transcriptional level, this is mediated by different RNA-binding regulators including YopD and CsrA, while the fate of mRNAs is balanced by ribonucleases. Here, we demonstrate that out of 11 tested putative RNases of Yersinia, two major RNases, the endoribonuclease RNase III, and the exonuclease and degradosome component PNPase play a crucial role in the activation of the Ysc-T3SS/Yop machinery. We show that they promote the decay of the lcrF mRNA encoding the common transcriptional activator LcrF of the Ysc-T3SS/Yop components. PNPase seems to act through the control of the effector YopD, known to promote the decay of the lcrF transcript. In contrast, RNase III triggers processes that reduce lcrF mRNA translation and stability, and involve CsrA. A transcriptome analysis further revealed that RNase III controls a series of events that include rapid and massive genetic reprogramming from mainly chromosomal-encoded genes to virulence-plasmid-encoded ysc-T3SS/yop genes. This control process does not only ensure immediate counter-measures during an immune attack, it also helps to overcome accompanying energetic and stress burdens and allows to rapidly readjust the genetic program after a successful defense.

microbiology↗

bioRxiv2023

DNA methylation profiling identifies TBKBP1 as potent amplifier of cytotoxic activity in CMV-specific human CD8+ T cells

Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. However, although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions when compared to bulk memory CD8+ T cells. Among them was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with TBKBP1 expression and was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies.

immunology↗

bioRxiv2022

Bursts in biosynthetic gene cluster transcription are accompanied by surges of natural compound production in the myxobacterium Sorangium sp.

We have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the production of known natural compounds, indicating that their biosynthesis was critially regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for myxobacterial producer strains, to boost compound yields by manipulating transcriptional activity.

genomics↗

bioRxiv2021

The thymic microenvironment gradually modulates the phenotype of thymus-homing peripheral conventional dendritic cells

Thymic conventional dendritic cells (t-DCs) are crucial for the development of T cells. A substantial fraction of t-DCs originates extrathymically and migrates to the thymus. Here, these cells contribute to key processes of central tolerance like the clonal deletion of self-reactive thymocytes and the generation of regulatory T (Treg) cells. So far, it is only incompletely understood which impact the thymic microenvironment has on thymus-homing conventional DCs (cDCs), which phenotypic changes occur after the entry of peripheral cDCs into the thymus and which functional properties these modulated cells acquire. In the present study, we mimicked the thymus-homing of peripheral cDCs by introducing ex vivo isolated splenic cDCs (sp-DCs) into re-aggregated thymic organ cultures (RTOCs). Already after two days of culture, the transcriptomic profile of sp-DCs was modulated and had acquired certain key signatures of t-DCs. The regulated genes included immunomodulatory cytokines and chemokines as well as co-stimulatory molecules. After four days of culture, sp-DCs appeared to have at least partially acquired the peculiar Treg cell-inducing capacity characteristic of t-DCs. Taken together, our findings indicate that peripheral cDCs possess a high degree of plasticity enabling them to quickly adapt to the thymus-specific microenvironment. We further provide indirect evidence that thymus-specific properties such as the efficient induction of Treg cells under homeostatic conditions can be partially transferred to thymus-homing peripheral cDC subsets.

immunology↗

bioRxiv2021

Postnatal lymph node expansion of stromal progenitors towards reticular and CD34+ stromal cell subsets is determined by distinct transcriptional programs

Gut-draining mesenteric lymph nodes (mLN) provide the framework and microenvironment to shape intestinal adaptive immune responses. We previously delineated transcriptional signatures in LN stromal cells (SC), pointing to tissue-specific variability in composition and immuno-modulatory function of SCs. Here, we dissect the tissue-specific epigenomic DNA accessibility and CpG methylation landscape of LN non-endothelial SCs and identify a microbiota-independent core epigenomic signature of LN SCs. By combined analysis of transcription factor (TF) binding sites together with the gene expression profiles of non-endothelial SCs, we delineated TFs poising skin-draining peripheral LN (pLN) SCs for pro-inflammatory responses. Furthermore, using scRNA-seq, we dissected the developmental trajectory of mLN SCs derived from postnatal to aged mice, identifying two distinct putative progenitors, namely CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors, which both feed the rapid postnatal LN expansion. Finally, we identified Irf3 as a key differentiation TF inferred from the epigenomic signature of mLN SCs that is dynamically expressed along the differentiation trajectories of FRCs, and validated Irf3 as a regulator of Cxcl9+ FRC differentiation. Together, our data constitute a comprehensive transcriptional and epigenomic map of mLN development and dissect location-specific, microbiota-independent properties of mLN non-endothelial SCs. As such, our findings represent a valuable resource to identify core transcriptional regulators that impinge on the developing mLN early in life, thereby shaping long-lasting intestinal adaptive immune responses.

immunology↗

bioRxiv2019

The alarmone (p)ppGpp is part of the heat shock response of Bacillus subtilis

Here, B. subtilis was used as a model organism to investigate how cells respond and adapt to proteotoxic stress conditions. Our experiments suggested that the stringent response, caused by raised levels of the (p)ppGpp alarmone, plays a role during thermotolerance development and the heat shock response. Accordingly, our experiments revealed a rapid increase of cellular (p)ppGpp levels upon heat shock as well as salt- and oxidative stress. Strains lacking (p)ppGpp exhibited increased stress sensitivity, while raised (p)ppGpp levels conferred increased stress tolerance to heat- and oxidative stress. During thermotolerance development, stress response genes were highly up-regulated together with a concurrent transcriptional down-regulation of the rRNA, which was influenced by the second messenger (p)ppGpp and the transcription factor Spx. Remarkably, we observed that (p)ppGpp appeared to control the cellular translational capacity and that during heat stress the raised cellular levels of the alarmone were able to curb the rate of protein synthesis. Furthermore, (p)ppGpp controls the heat-induced expression of Hpf and thus the formation of translationally inactive 100S disomes. These results indicate that B. subtilis cells respond to heat-mediated protein unfolding and aggregation, not only by raising the cellular repair capacity, but also by decreasing translation involving (p)ppGpp mediated stringent response to concurrently reduce the protein load for the cellular protein quality control system.\n\nAuthor SummaryHere we demonstrate that the bacterial stringent response, which is known to slow down translation upon sensing nutrient starvation, is also intricately involved in the stress response of B. subtilis cells. The second messengers (p)ppGpp act as pleiotropic regulators during the adaptation to heat stress. (p)ppGpp slows down translation and is also involved in the transcriptional down-regulation of the translation machinery, together with the transcriptional stress regulator Spx. The stress-induced elevation of cellular (p)ppGpp levels confers increased stress tolerance and facilitates an improved protein homeostasis by reducing the load on the protein quality control system.

microbiology↗

bioRxiv2017

Critical Assessment of Metagenome Interpretation - a benchmark of computational metagenomics software

In metagenome analysis, computational methods for assembly, taxonomic profiling and binning are key components facilitating downstream biological data interpretation. However, a lack of consensus about benchmarking datasets and evaluation metrics complicates proper performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on datasets of unprecedented complexity and realism. Benchmark metagenomes were generated from ~700 newly sequenced microorganisms and ~600 novel viruses and plasmids, including genomes with varying degrees of relatedness to each other and to publicly available ones and representing common experimental setups. Across all datasets, assembly and genome binning programs performed well for species represented by individual genomes, while performance was substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below the family level. Parameter settings substantially impacted performances, underscoring the importance of program reproducibility. While highlighting current challenges in computational metagenomics, the CAMI results provide a roadmap for software selection to answer specific research questions.

bioinformatics↗