scholarly journals Ser/Thr phospho-regulation by PknB and Stp mediates bacterial quiescence and antibiotic persistence in Staphylococcus aureus

2021 ◽  
Author(s):  
Markus Huemer ◽  
Srikanth Mairpady Shambat ◽  
Sandro Pereira ◽  
Lies Van Gestel ◽  
Judith Bergada-Pijuan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ribosomal Proteins ◽  
Environmental Changes ◽  
Acid Stress ◽  
Elongation Factor ◽  
Growth Delay ◽  
Lag Phase ◽  
Cellular Processes ◽  
Translational Activity

Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Most antibiotics act on metabolically active bacteria in order to eradicate them. Thus, bacteria with minimized energy consumption resulting in metabolic quiescence, have increased tolerance to antibiotics. The most energy intensive process in cells - protein synthesis - is attenuated in bacteria entering into quiescence. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) can fine-tune essential cellular processes, thereby enabling bacteria to quickly respond to environmental changes and to modulate quiescence. Here, we show that deletion of the only annotated functional STP, named Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity under different stress challenges, including acidic pH, intracellular milieu and in vivo abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins including the essential translation elongation factor EF-G. Finally, we show that acid stress leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlating with increased antibiotic persistence.

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

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.

Renal epithelial gene expression profile and bismuth-induced resistance against cisplatin nephrotoxicity

2003 ◽  
Vol 22 (10) ◽  
pp. 535-540 ◽  
Author(s):  
Berend T Leussink ◽  
Hans J Baelde ◽  
Thirza M Broekhuizen-van den Berg ◽  
Emile de Heer ◽  
Gijsbert B van der Voet ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Animal Experiments ◽  
Elongation Factor ◽  
Limiting Factor ◽  
Bismuth Salts ◽  
Proximal Tubular ◽  
A Cell ◽  
Induced Apoptosis

Nephrotoxicity is the most important dose-limiting factor in cisplatin based anti-neoplastic treatment. Pretreatment with bismuth salts, used as pharmaceuticals to treat gastric disorders, has been demonstrated to reduce cisplatin-induced renal cell death in clinical settings and during in vivo and in vitro animal experiments. To investigate the genomic basis of this renoprotective effect, we exposed NRK-52E cells, a cell line of rat proximal tubular epithelial origin, to 33 mM Bi3 for 12 hours, which made them resistant to cisplatin-induced apoptosis. Differentially expressed genes in treated and untreated NRK-52E cells were detected by subtraction PCR and microarray techniques. Genes found to be down regulated (0.17 / 0.31-times) were cytochrome c oxidase subunit I, BAR (an apoptosis regulator), heat-shock protein 70-like protein, and three proteins belonging to the translation machinery (ribosomal proteins S7 and L17, and S1, a member of the elongation factor 1-alpha family). The only up-regulated gene was glutathione Stransferase subunit 3A (1.89-times). Guided by the expression levels of these genes, it may be possible to improve renoprotective treatments during anti-neoplastic therapies.

Raf kinases in signal transduction and interaction with translation machinery

2013 ◽  
Vol 4 (4) ◽  
pp. 391-399 ◽  
Author(s):  
Nunzia Migliaccio ◽  
Carmen Sanges ◽  
Immacolata Ruggiero ◽  
Nicola M. Martucci ◽  
Emilia Rippa ◽  
...  
Keyword(s):  
Translational Control ◽  
Elongation Factor ◽  
Cancer Cell Line ◽  
Pi3k Pathway ◽  
Cancer Tissue ◽  
Translational Machinery ◽  
Cellular Processes ◽  
Survival Pathway

AbstractIn recent years, a large amount of evidence has given a central role to translational control in diseases such as cancer, tissue hypertrophy and neurodegeneration. Its deregulation can directly modulate cell cycling, transformation and survival response. The aim of this review is to describe the interaction between Raf activation and the main characters of the translational machinery, such as the elongation factor 1A (eEF1A), which has been recognized in recent years as one of the most interesting putative oncogenes. A particular emphasis is given to an intriguing non-canonical role that eEF1A can play in the relationship between the Ras→Raf-1→MEK1→ERK-1/2 and PI3K→Akt signaling pathways. Recently, our group has described a C-Raf kinase-mediated phosphorylation of eEF1A triggered by a survival pathway induced upon interferon alpha (IFNα) treatment in the human epidermoid cancer cell line (H1355). This phosphorylation seems to be the center of the survival pathway that counteracts the well-known pro-apoptotic function of IFNα. Furthermore, we have identified two new phosphorylation sites on eEF1A (Ser21 and Thr88) that are substrates for Raf kinases in vitro and, likely, in vivo as well. These residues seem to have a significant functional role in the control of cellular processes, such as cell proliferation and survival. In fact, overexpression of eEF1A2 in gemcitabine-treated cancer cells caused the upregulation of phosphoAkt and an increase in cell viability, thereby suggesting that eEF1A2 could exert its oncogenic behavior by participating in the regulation of PI3K pathway.

scholarly journals Bmh1p (14-3-3) mediates pathways associated with virulence in Candida albicans

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1536-1546 ◽  
Author(s):  
Michelle N. Kelly ◽  
Douglas A. Johnston ◽  
Bethany A. Peel ◽  
Timothy W. Morgan ◽  
Glen E. Palmer ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mouse Model ◽  
Virulence Factors ◽  
Environmental Changes ◽  
Pathogenic Fungus ◽  
Binding Pocket ◽  
Lag Phase ◽  
Models Of Disease

The ability of the pathogenic fungus Candida albicans to cause disease requires rapid adaptation to changes in the host environment and to an evolving host immune response. The identification of ‘virulence factors’ using in vitro characterization of mutant strains has traditionally relied on a common set of phenotypic and biochemical assays (most often performed at 30 °C) and the subsequent correlation with their corresponding virulence in mouse models of disease. Utilizing a panel of isogenic mutants for the multifunctional signal-modulating 14-3-3 protein (Bmh1p), we have found that specific mutations affect a variety of different pathways currently associated with virulence, including those involved with the formation of filaments, as well as interaction with host immune cells. Surprisingly, our studies revealed that deficiencies in many of these pathways do not always correlate with virulence in a mouse model of disseminated infection. Mutations within the binding pocket of Bmh1p that affect the ability of the protein to efficiently bind ligand had varying effects on the results of a number of in vitro and in vivo assays. The capability, in vitro, to filament in embedment conditions, and to filament and form chlamydospores under microaerophilic conditions on cornmeal agar, does not correlate with virulence. It is likely that only a subset of hyphal signalling pathways is actually required for the establishment of infection in the disseminated mouse model. Most importantly, our results suggest that the delayed onset of lag-phase growth in vitro at 37 °C, and not at 30 °C, results in an inability of these mutants to rapidly adjust to environmental changes in vivo and may be responsible for their increased clearance and reduced virulence. It is critical, therefore, that future in vitro studies of putative virulence factors in C. albicans include careful characterization at physiological temperatures.

scholarly journals Allomorphy as a mechanism of post-translational control of enzyme activity

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Henry P. Wood ◽  
F. Aaron Cruz-Navarrete ◽  
Nicola J. Baxter ◽  
Clare R. Trevitt ◽  
Angus J. Robertson ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Translational Control ◽  
Environmental Changes ◽  
Control Mechanism ◽  
Enzyme Regulation ◽  
Peptide Bond ◽  
Lag Phase ◽  
Phosphoryl Transfer ◽  
Fine Control

Abstract Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

scholarly journals Yeast Asc1p and Mammalian RACK1 Are Functionally Orthologous Core 40S Ribosomal Proteins That Repress Gene Expression

2004 ◽  
Vol 24 (18) ◽  
pp. 8276-8287 ◽  
Author(s):  
Vincent R. Gerbasi ◽  
Connie M. Weaver ◽  
Salisha Hill ◽  
David B. Friedman ◽  
Andrew J. Link
Keyword(s):  
Gene Expression ◽  
Translational Control ◽  
Ribosomal Proteins ◽  
Translational Activity ◽  
Eukaryotic Gene ◽  
Associated Proteins ◽  
Yeast Ribosomes ◽  
Specific Proteins ◽  
Conserved Core

ABSTRACT Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Δ null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.

scholarly journals Antibacterial apple cider vinegar eradicates methicillin resistant Staphylococcus aureus and resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Darshna Yagnik ◽  
Malcolm Ward ◽  
Ajit J. Shah
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Ribosomal Proteins ◽  
Elongation Factor ◽  
Phosphoglycerate Kinase ◽  
Resistant Bacteria ◽  
High Morbidity ◽  
Label Free ◽  
Methicillin Resistant ◽  
Apple Cider

AbstractMethicillin-resistant Staphylococcus aureus (MRSA) and resistant Escherichia coli (rE.coli) infections can spread rapidly. Further they are associated with high morbidity and mortality from treatment failure. Therapy involves multiple rounds of ineffective antibiotics alongside unwanted side effects, alternative treatments are crucial. Apple cider vinegar (ACV) is a natural, vegan product that has been shown to have powerful antimicrobial activity hence we investigated whether ACV could ameliorate these resistant bacteria. The minimum dilution of ACV required for growth inhibition was comparable for both bacteria (1/25 dilution of ACV liquid and ACV tablets at 200 µg/ml were effective against rE. coli and MRSA). Monocyte co-culture with microbes alongside ACV resulted in an increase in monocyte phagocytosis by 21.2% and 33.5% compared to non-ACV treated but MRSA or rE. coli stimulated monocytes, respectively. Label free quantitative proteomic studies of microbial protein extracts demonstrated that ACV penetrated microbial cell membranes and organelles, altering the expression of key proteins. This resulted in significant reductions in total protein expression, moreover we could only detect ribosomal proteins; 50 s 30 s, enolase, phosphenol pyruvate and the ATP synthase subunit in rE. coli. Elongation factor iNOS and phosphoglycerate kinase OS were the only proteins present in MRSA samples following ACV treatment.

scholarly journals Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

2014 ◽  
Vol 289 (44) ◽  
pp. 30499-30510 ◽  
Author(s):  
Erna Davydova ◽  
Angela Y. Y. Ho ◽  
Jedrzej Malecki ◽  
Anders Moen ◽  
Jorrit M. Enserink ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Sequence Similarity ◽  
Elongation Factor ◽  
Protein Translation ◽  
Cellular Protein ◽  
Yeast Cells ◽  
Lysine Methylation ◽  
Elongation Factor 2

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.

scholarly journals Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

1998 ◽  
Vol 331 (2) ◽  
pp. 423-430 ◽  
Author(s):  
Monika ÜHLEIN ◽  
Wolfgang WEGLÖHNER ◽  
Henning URLAUB ◽  
Brigitte WITTMANN-LIEBOLD
Keyword(s):  
Ribosomal Proteins ◽  
Protein Biosynthesis ◽  
Complex Structure ◽  
Peptide Bond ◽  
Histidine Residue ◽  
Peptide Bond Formation ◽  
Homologous Proteins ◽  
Translational Activity ◽  
Translational Apparatus

The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In vivo replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and archaebacterial ribosomes. These hybrid ribosomes are active in protein biosynthesis, as proven by polysome analysis and poly(U)-dependent polyphenylalanine synthesis. Furthermore, we demonstrate that a specific, highly conserved, histidine residue in the C-terminal region of L2 is essential for the function of the translational apparatus. Replacement of this histidine residue in the human and archaebacterial proteins by glycine, arginine or alanine had no effect on ribosome assembly, but strongly reduced the translational activity of ribosomes containing these mutants.

Microtubules: greater than the sum of the parts

2013 ◽  
Vol 41 (6) ◽  
pp. 1736-1744 ◽  
Author(s):  
Jonathan L.D. Lawson ◽  
Rafael E. Carazo Salas
Keyword(s):  
Environmental Changes ◽  
Super Resolution ◽  
Integrative Approach ◽  
Microtubule Cytoskeleton ◽  
Cellular Processes ◽  
Interdisciplinary Approaches ◽  
High Degree ◽  
Super Resolution Microscopy ◽  
Shed Light

The post-genomic era has produced a variety of new investigation technologies, techniques and approaches that may offer exciting insights into many long-standing questions of scientific research. The microtubule cytoskeleton is a highly conserved system that shows a high degree of internal complexity, is known to be integral to many cell systems and functions on a fundamental level. After decades of study, much is still unknown about microtubules in vivo from the control of dynamics in living cells to their responses to environmental changes and responses to other cellular processes. In the present article, we examine some outstanding questions in the microtubule field and propose a combination of emerging interdisciplinary approaches, i.e. high-throughput functional genomics techniques, quantitative and super-resolution microscopy, and in silico modelling, that could shed light on the systemic regulation of microtubules in cells by networks of regulatory factors. We propose that such an integrative approach is key to elucidate the function of the microtubule cytoskeleton as a complete responsive integral biological system.

Sign in / Sign up

Export Citation Format

Share Document