Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin

Abstract A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin–antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.

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Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions

Nucleic Acids Research ◽

10.1093/nar/gkaa404 ◽

2020 ◽

Vol 48 (12) ◽

pp. e71-e71 ◽

Cited By ~ 1

Author(s):

Christian Twittenhoff ◽

Vivian B Brandenburg ◽

Francesco Righetti ◽

Aaron M Nuss ◽

Axel Mosig ◽

...

Keyword(s):

High Throughput Sequencing ◽

Yersinia Pseudotuberculosis ◽

Structural Information ◽

Dimethyl Sulfate ◽

Bacterial Pathogen ◽

Structural Features ◽

Global Scale ◽

Rna Molecules ◽

Different Temperatures

Abstract The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed ‘Lead-seq’, provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5′-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.

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Advanced Resistance Studies Identify Two Discrete Mechanisms in Staphylococcus aureus to Overcome Antibacterial Compounds that Target Biotin Protein Ligase

Antibiotics ◽

10.3390/antibiotics9040165 ◽

2020 ◽

Vol 9 (4) ◽

pp. 165 ◽

Cited By ~ 1

Author(s):

Andrew J. Hayes ◽

Jiulia Satiaputra ◽

Louise M. Sternicki ◽

Ashleigh S. Paparella ◽

Zikai Feng ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Transcriptional Repression ◽

Molecular Mechanisms ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Resistance Rate ◽

Biotin Protein Ligase ◽

Essential Enzyme

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

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Role of GADD153 (growth arrest- and DNA damage-inducible gene 153) in vascular smooth muscle cell apoptosis

Clinical Science ◽

10.1042/cs1000275 ◽

2001 ◽

Vol 100 (3) ◽

pp. 275-281 ◽

Cited By ~ 11

Author(s):

Michiya IGASE ◽

Takafumi OKURA ◽

Michitsugu NAKAMURA ◽

Yasunori TAKATA ◽

Yutaka KITAMI ◽

...

Keyword(s):

Dna Damage ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Molecular Mechanisms ◽

Growth Arrest ◽

Cell Contact ◽

Inducible Gene

GADD153 (growth arrest- and DNA damage-inducible gene 153) is expressed at very low levels in growing cells, but is markedly induced in response to a variety of cellular stresses, including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations. Forced expression of GADD153 induces cell cycle arrest in many types of cells. It is also reported that GADD153 is directly associated with apoptosis. Recently we have reported that platelet-derived growth factor (PDGF)-BB induces apoptosis in cultured vascular smooth muscle cells (VSMC), but only when 100% confluency is reached. These results suggested that cell–cell contact inhibition (cell growth arrest) may be a critical factor for induction of VSMC apoptosis by PDGF-BB. In the present study, we explored the role of GADD153, one of a number of growth-arrest-related gene products, in the molecular mechanisms of VSMC apoptosis in vitro and in vivo. GADD153 was markedly induced at both the mRNA and protein levels, in parallel with the induction of VSMC apoptosis, after treatment with PDGF-BB. Moreover, overexpression of GADD153 in VSMC significantly reduced cell viability and induced apoptosis. In the carotid artery balloon injury model in rats, GADD153 protein was expressed in apoptotic VSMC which were positively stained by in situ DNA labelling. These results demonstrate an important role for GADD153 in the molecular mechanisms of VSMC apoptosis.

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Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis

10.1101/847723 ◽

2019 ◽

Author(s):

E. P. Kusnadi ◽

A. S. Trigos ◽

C. Cullinane ◽

D. L. Goode ◽

O. Larsson ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Rational Design ◽

Molecular Mechanisms ◽

Single Agent ◽

Acquired Resistance ◽

Cellular Metabolism ◽

Mrna Translation ◽

Pol I ◽

Polymerase I

AbstractElevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA Polymerase I (Pol I) transcription, revealed single agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here we show that this improved efficacy is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this co-treatment is driven by translational re-wiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies identify the molecular mechanisms underpinning the response of blood cancers to selective ribosome biogenesis inhibitors and identify metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

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Transcriptome profiling of mastitis-specialized Staphylococcus aureus reveals the impact of low-oxygenation on the regulation of unique pawthways after internalization into bovine mammary alveolar cell-T (MAC-T)

10.21203/rs.2.17396/v1 ◽

2019 ◽

Author(s):

Kamaleldin B Said ◽

Xin Zhao ◽

Marcus B Jones ◽

Rosslyn Maybank ◽

Scott Peterson

Keyword(s):

Staphylococcus Aureus ◽

Molecular Mechanisms ◽

Protein A ◽

Transcriptome Profiling ◽

Low Oxygen ◽

Gene Expressions ◽

Alveolar Cell ◽

Oxygen Levels ◽

The Impact

Abstract Background Mastitis-specialized lineages of Staphylococcus aureus are important pathogens in the dairy industry. The molecular mechanisms underlying host- and organ-specialization in these lineages are still not fully understood. Recent findings suggested that differential expression of genes may have contributed to the evolution of strains with enhanced virulence. However, studies on gene expressions under key intra-mammary conditions are quite limited for mastitis S. aureus . The purpose of the study was to investigate the influence of low oxygen levels on the transcriptome profiles of bovine matitis S. aureus , using high-throughput whole genome qRT-PCR.Results Results showed that under normal oxygenation, a mastitis-isolate expressed subsets of genes for adaptation, environmental-sensing, and binding including merR, sigB , vraS , yycG/yycF , araC , and tetR . In addition, coupling of fermentative metabolism to virulence was indicated by accumulated transcripts for catabolite control protein A ( ccpA) and pentose-monophosphate operon and depleted transcripts for tricaroxylic acid cycle. Furthermore, sarU mediated agr activation was evidented by transcripts for toxins, adaptation, and in-vivo viability factors as staphopains and gntR operon. On the other hand, reduced oxygenation increased transcription of fibrinogen-binding genes, isd- operon, and sdrH showing aggressive adherence phenotype. While normal oxygenation produced gene activities for quick and aggressive responses, low-oxygenation induced phenotypes for persistence, binding, and metabolic inactivity.Conclusion Significant differences in the transcriptional profiles were observed for mammary alveolar cell-T (MAC-T) internalized S. aureus under low oxygen levels compared to that at normal levels. This indicated that low oxygen is an important key mammary factor that influence transcriptome profiles of intra-mammary-specific phenotypes of S. aureus . These findings will help in understanding the effect of oxygen on the differentiation and evolution of intramammary S. aureus .

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Effects of Peptide Thanatin on the Growth and Transcriptome of Penicillium digitatum

Frontiers in Microbiology ◽

10.3389/fmicb.2020.606482 ◽

2020 ◽

Vol 11 ◽

Author(s):

Guirong Feng ◽

Xindan Li ◽

Wenjun Wang ◽

Lili Deng ◽

Kaifang Zeng

Keyword(s):

Ribosome Biogenesis ◽

Molecular Mechanisms ◽

High Efficiency ◽

Citrus Fruit ◽

Underlying Mechanism ◽

Treated Group ◽

Penicillium Digitatum ◽

Major Facilitator Superfamily

Penicillium digitatum is the most damaging pathogen provoking green mold in citrus fruit during storage, and there is an urgent need for novel antifungal agents with high efficiency. The aim of this study was to investigate the antifungal effects of peptide thanatin against P. digitatum and the molecular mechanisms. Results showed that peptide thanatin had a prominent inhibitory effect on P. digitatum by in vitro and in vivo test. A total of 938 genes, including 556 downregulated and 382 upregulated genes, were differentially expressed, as revealed by RNA-seq of whole P. digitatum genomes analysis with or without thanatin treatment. The downregulated genes mainly encoded RNA polymerase, ribosome biogenesis, amino acid metabolism, and major facilitator superfamily. The genes associated with heat shock proteins and antioxidative systems were widely expressed in thanatin-treated group. DNA, RNA, and the protein content of P. digitatum were significantly decreased after thanatin treatment. In conclusion, thanatin could inhibit the growth of P. digitatum, and the underlying mechanism might be the genetic information processing and stress response were affected. The research will provide more precise and directional clues to explore the inhibitory mechanism of thanatin on growth of P. digitatum.

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Molecular reprogramming and phenotype switching in Staphylococcus aureus lead to high antibiotic persistence and affect therapy success

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014920118 ◽

2021 ◽

Vol 118 (7) ◽

pp. e2014920118

Author(s):

Markus Huemer ◽

Srikanth Mairpady Shambat ◽

Judith Bergada-Pijuan ◽

Sandra Söderholm ◽

Mathilde Boumasmoud ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Ribosomal Proteins ◽

Molecular Mechanisms ◽

Prolonged Treatment ◽

Infection Model ◽

Lag Phase ◽

Recurrent Infections ◽

Surgical Interventions

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.

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Sp1-Mediated circRNA circHipk2 Regulates Myogenesis by Targeting Ribosomal Protein Rpl7

Genes ◽

10.3390/genes12050696 ◽

2021 ◽

Vol 12 (5) ◽

pp. 696

Author(s):

Junyu Yan ◽

Yalan Yang ◽

Xinhao Fan ◽

Yijie Tang ◽

Zhonglin Tang

Keyword(s):

Ribosomal Protein ◽

Ribosome Biogenesis ◽

C2c12 Cells ◽

Circular Rnas ◽

Exon 2 ◽

Rna Molecules ◽

Assembly Factor ◽

And Function

Circular RNAs (circRNAs) represent a class of covalently closed single-stranded RNA molecules that are emerging as essential regulators of various biological processes. The circRNA circHipk2 originates from exon 2 of the Hipk2 gene in mice and was reported to be involved in acute promyelocytic leukemia and myocardial injury. However, the functions and mechanisms of circHipk2 in myogenesis are largely unknown. Here, to deepen our knowledge about the role of circHipk2, we studied the expression and function of circHipk2 during skeletal myogenesis. We found that circHipk2 was mostly distributed in the cytoplasm, and dynamically and differentially expressed in various myogenesis systems in vitro and in vivo. Functionally, overexpression of circHipk2 inhibited myoblast proliferation and promoted myotube formation in C2C12 cells, whereas the opposite effects were observed after circHipk2 knockdown. Mechanistically, circHipk2 could directly bind to ribosomal protein Rpl7, an essential 60S preribosomal assembly factor, to inhibit ribosome translation. In addition, we verified that transcription factor Sp1 directly bound to the promoter of circHipk2 and affected the expression of Hipk2 and circHipk2 in C2C12 myoblasts. Collectively, these findings identify circHipk2 as a candidate circRNA regulating ribosome biogenesis and myogenesis proliferation and differentiation.

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