scholarly journals NrnA is a linear dinucleotide phosphodiesterase with limited function in cyclic dinucleotide metabolism in Listeria monocytogenes

2021 ◽  
Author(s):  
Aaron R. Gall ◽  
Brian Y. Hsueh ◽  
Cheta Siletti ◽  
Christopher M. Waters ◽  
TuAnh N. Huynh
Keyword(s):  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Cell Infection ◽  
Cellular Processes ◽  
Hydrolytic Activities ◽  
Limited Function ◽  
Cyclic Dinucleotides ◽  
Virulence Regulator ◽  
Cyclic Dinucleotide

Listeria monocytogenes produces both c-di-AMP and c-di-GMP to mediate many important cellular processes, but the levels of both nucleotides must be regulated. C-di-AMP accumulation attenuates virulence and diminishes stress response, and c-di-GMP accumulation impairs bacterial motility. An important regulatory mechanism to maintain c-di-AMP and c-di-GMP homeostasis is to hydrolyze them to the linear dinucleotides pApA and pGpG, respectively, but the fates of these hydrolytic products have not been examined in L. monocytogenes . We found that NrnA, a stand-alone DHH-DHHA1 phosphodiesterase, has a broad substrate range, but with a strong preference for linear dinucleotides over cyclic dinucleotides. Although NrnA exhibited detectable cyclic dinucleotide hydrolytic activities in vitro, NrnA had negligible effects on their levels in the bacterial cell, even in the absence of the c-di-AMP phosphodiesterases PdeA and PgpH. The Δ nrnA mutant had a mammalian cell infection defect that was fully restored by E. coli Orn. Together, our data indicate that L. monocytogenes NrnA is functionally orthologous to Orn, and its preferred physiological substrates are most likely linear dinucleotides. Furthermore, our findings revealed that, unlike some other c-di-AMP and c-di-GMP-producing bacteria, L. monocytogenes does not employ their hydrolytic products to regulate their phosphodiesterases, at least at the pApA and pGpG levels in the Δ nrnA mutant. Finally, the Δ nrnA infection defect was overcome by constitutive activation of PrfA, the master virulence regulator, suggesting that accumulated linear dinucleotides might inhibit the expression, stability, or function of PrfA-regulated virulence factors. IMPORTANCE Listeria monocytogenes produces both c-di-AMP and c-di-GMP, and encodes specific phosphodiesterases that degrade them into pApA and pGpG, respectively, but the metabolism of these products has not been characterized in this bacterium. We found that L. monocytogenes NrnA degrades a broad range of nucleotides. Among the tested cyclic and linear substrates, it exhibits a strong biochemical and physiological preference the linear dinucleotides pApA, pGpG, and pApG. Unlike in some other bacteria, these oligoribonucleotides do not appear to interfere with cyclic dinucleotide hydrolysis. The absence of NrnA is well tolerated by L. monocytogenes in broth cultures but impairs its ability to infect mammalian cells. These findings indicate a separation of cyclic dinucleotide signaling and oligoribonucleotide metabolism in L. monocytogenes .

scholarly journals NrnA is a linear dinucleotide phosphodiesterase with limited function in cyclic dinucleotide metabolism in Listeria monocytogenes

2021 ◽  
Author(s):  
Aaron R. Gall ◽  
Cheta Siletti ◽  
TuAnh N. Huynh
Keyword(s):  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Cell Infection ◽  
Cellular Processes ◽  
Hydrolytic Activities ◽  
Limited Function ◽  
Cyclic Dinucleotides ◽  
Virulence Regulator ◽  
Cyclic Dinucleotide

ABSTRACTListeria monocytogenes produces both c-di-AMP and c-di-GMP to mediate many important cellular processes, but the levels of both nucleotides must be regulated. C-di-AMP accumulation attenuates virulence and diminishes stress response, and c-di-GMP accumulation impairs bacterial motility. An important regulatory mechanism to maintain c-di-AMP and c-di-GMP homeostasis is to hydrolyze them to the linear dinucleotides pApA and pGpG, respectively, but the fates of these hydrolytic products have not been examined in L. monocytogenes. We found that NrnA, a stand-alone DHH-DHHA1 phosphodiesterase, has a broad substrate range, but with a strong preference for linear dinucleotides over cyclic dinucleotides. Although NrnA exhibited detectable cyclic dinucleotide hydrolytic activities in vitro, NrnA had negligible effects on their levels in the bacterial cell, even in the absence of the c-di-AMP phosphodiesterases PdeA and PgpH. The ΔnrnA mutant had a mammalian cell infection defect that was fully restored by E. coli Orn. Together, our data indicate that L. monocytogenes NrnA is functionally orthologous to Orn, and its preferred physiological substrates are most likely linear dinucleotides. Furthermore, our findings revealed that, unlike some other c-di-AMP and c-di-GMP-producing bacteria, L. monocytogenes does not employ their hydrolytic products to regulate their phosphodiesterases, at least at the pApA and pGpG levels in the ΔnrnA mutant. Finally, the ΔnrnA infection defect was overcome by constitutive activation of PrfA, the master virulence regulator, suggesting that accumulated linear dinucleotides might inhibit the expression, stability, or function of PrfA-regulated virulence factors.IMPORTANCEListeria monocytogenes produces both c-di-AMP and c-di-GMP, and encodes specific phosphodiesterases that degrade them into pApA and pGpG, respectively, but the metabolism of these products has not been characterized in this bacterium. We found that L. monocytogenes harbors an NrnA homolog that degrades a broad range of nucleotides, but exhibits a strong biochemical and physiological preference for linear dinucleotides, including pApA and pGpG. Unlike in some other bacteria, these oligoribonucleotides do not appear to interfere with cyclic dinucleotide hydrolysis. The absence of NrnA is well tolerated by L. monocytogenes in broth cultures but impairs its ability to infect mammalian cells. These findings indicate a separation of cyclic dinucleotide signaling and oligoribonucleotide metabolism in L. monocytogenes.

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

scholarly journals Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species

2015 ◽  
Vol 198 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Natalia Tschowri
Keyword(s):  
Cell Wall ◽  
Current Knowledge ◽  
Second Messengers ◽  
Streptomyces Species ◽  
Content Type ◽  
Regulatory Pathways ◽  
Cellular Processes ◽  
Signal Transduction Networks ◽  
Cyclic Dinucleotides ◽  
Cyclic Dinucleotide

The cyclic dinucleotides cyclic 3′,5′-diguanylate (c-di-GMP) and cyclic 3′,5′-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling inStreptomycesspecies and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria.

scholarly journals Human Monocyte Recognition of Adenosine-Based Cyclic Dinucleotides Unveils the A2a GαsProtein-Coupled Receptor Tonic Inhibition of Mitochondrially Induced Cell Death

2014 ◽  
Vol 35 (2) ◽  
pp. 479-495 ◽  
Author(s):  
Marie Tosolini ◽  
Frédéric Pont ◽  
Delphine Bétous ◽  
Emmanuel Ravet ◽  
Laetitia Ligat ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Mitochondrial Permeability Transition ◽  
Human Monocyte ◽  
Inverse Agonist ◽  
Apoptotic Pathway ◽  
Cyclic Dinucleotides ◽  
Agonist Ligands

Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cellsin vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a Gαs-coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic.In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34+cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a Gαsprotein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death.

scholarly journals Detoxification of methylglyoxal by the glyoxalase system is required for glutathione availability and virulence activation in Listeria monocytogenes

2021 ◽  
Vol 17 (8) ◽  
pp. e1009819
Author(s):  
Andrea Anaya-Sanchez ◽  
Ying Feng ◽  
John C. Berude ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Transcriptional Activation ◽  
Protein Glycation ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Cell Monolayers ◽  
Food Borne ◽  
Virulence Regulator ◽  
Allosteric Activator

Listeria monocytogenes is a Gram-positive, food-borne pathogen that lives a biphasic lifestyle, cycling between the environment and as a facultative intracellular pathogen of mammals. Upon entry into host cells, L. monocytogenes upregulates expression of glutathione synthase (GshF) and its product, glutathione (GSH), which is an allosteric activator of the master virulence regulator PrfA. Although gshF mutants are highly attenuated for virulence in mice and form very small plaques in host cell monolayers, these virulence defects can be fully rescued by mutations that lock PrfA in its active conformation, referred to as PrfA*. While PrfA activation can be recapitulated in vitro by the addition of reducing agents, the precise biological cue(s) experienced by L. monocytogenes that lead to PrfA activation are not known. Here we performed a genetic screen to identify additional small-plaque mutants that were rescued by PrfA* and identified gloA, which encodes glyoxalase A, a component of a GSH-dependent methylglyoxal (MG) detoxification system. MG is a toxic byproduct of metabolism produced by both the host and pathogen, which if accumulated, causes DNA damage and protein glycation. As a facultative intracellular pathogen, L. monocytogenes must protect itself from MG produced by its own metabolic processes and that of its host. We report that gloA mutants grow normally in broth, are sensitive to exogenous MG and severely attenuated upon IV infection in mice, but are fully rescued for virulence in a PrfA* background. We demonstrate that transcriptional activation of gshF increased upon MG challenge in vitro, and while this resulted in higher levels of GSH for wild-type L. monocytogenes, the glyoxalase mutants had decreased levels of GSH, presumably due to the accumulation of the GSH-MG hemithioacetal adduct. These data suggest that MG acts as a host cue that leads to GSH production and activation of PrfA.

scholarly journals The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo.

1996 ◽  
Vol 183 (2) ◽  
pp. 359-369 ◽  
Author(s):  
J L Gaillard ◽  
F Jaubert ◽  
P Berche
Keyword(s):  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Defense Mechanisms ◽  
Inflammatory Cells ◽  
Cell Infection ◽  
Chromosomal Locus ◽  
Direct Role ◽  
Innate Defense ◽  
A Cell

The intracellular parasite Listeria monocytogenes is able to induce its internalization by cultured mammalian cells that are not normally phagocytic. This process requires the expression of the chromosomal locus inlAB. We studied the virulence of an inlAB mutant and of its parent in murine listeriosis. Irrespective of the route of inoculation, the inlAB mutant was severely attenuated for growth in the liver. The livers of mice inoculated with the inlAB mutant displayed much smaller infectious foci than the parent as early as 24 h after infection. Electron microscopy showed that these foci consisted of a few inflammatory cells, with few bacteria; bacteria were rarely found within hepatocytes. In contrast, foci in livers of mice inoculated with the parent consisted of islets of heavily infected hepatocytes that were infiltrated by numerous neutrophils; bacteria seemed intact within hepatocytes and damaged within neutrophils. A direct role of inlAB for the entry of L. monocytogenes into hepatocytes was confirmed in a cell infection system using the murine embryonic hepatocyte cell line TIB73. The inlAB mutant was approximately 20-fold less invasive in trans. The "invasion locus" inlAB contributes to protect L. monocytogenes from the host's innate defense mechanisms by promoting its entry into hepatocytes.

scholarly journals CodY-Mediated c-di-GMP-Dependent Inhibition of Mammalian Cell Invasion in Listeria monocytogenes

2017 ◽  
Vol 200 (5) ◽  
Author(s):  
Ahmed M. Elbakush ◽  
Kurt W. Miller ◽  
Mark Gomelsky
Keyword(s):  
Transcription Factor ◽  
Listeria Monocytogenes ◽  
Mammalian Cell ◽  
Cell Invasion ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Second Messenger ◽  
Content Type ◽  
Branched Chain

ABSTRACT Elevated levels of the second messenger c-di-GMP suppress virulence in diverse pathogenic bacteria, yet mechanisms are poorly characterized. In the foodborne pathogen Listeria monocytogenes , high c-di-GMP levels inhibit mammalian cell invasion. Here, we show that invasion is impaired because of the decreased expression levels of internalin genes whose products are involved in invasion. We further show that at high c-di-GMP levels, the expression of the entire virulence regulon is suppressed, and so is the expression of the prfA gene encoding the master activator of the virulence regulon. Analysis of mechanisms controlling prfA expression pointed to the transcription factor CodY as a c-di-GMP-sensitive component. In high-c-di-GMP strains, codY gene expression is decreased, apparently due to the lower activity of CodY, which functions as an activator of codY transcription. We found that listerial CodY does not bind c-di-GMP in vitro and therefore investigated whether c-di-GMP levels affect two known cofactors of listerial CodY, branched-chain amino acids and GTP. Our manipulation of branched-chain amino acid levels did not perturb the c-di-GMP effect; however, our replacement of listerial CodY with the streptococcal CodY homolog, whose activity is GTP independent, abolished the c-di-GMP effect. The results of this study suggest that elevated c-di-GMP levels decrease the activity of the coordinator of metabolism and virulence, CodY, possibly via lower GTP levels, and that decreased CodY activity suppresses L. monocytogenes virulence by the decreased expression of the PrfA virulence regulon. IMPORTANCE Listeria monocytogenes is a pathogen causing listeriosis, a disease responsible for the highest mortality rate among foodborne diseases. Understanding how the virulence of this pathogen is regulated is important for developing treatments to decrease the frequency of listerial infections in susceptible populations. In this study, we describe the mechanism through which elevated levels of the second messenger c-di-GMP inhibit listerial invasion in mammalian cells. Inhibition is caused by the decreased activity of the transcription factor CodY that coordinates metabolism and virulence.

scholarly journals The regulatory enzymes and protein substrates for the lysine β-hydroxybutyrylation pathway

2021 ◽  
Vol 7 (9) ◽  
pp. eabe2771 ◽  
Author(s):  
He Huang ◽  
Di Zhang ◽  
Yejing Weng ◽  
Kyle Delaney ◽  
Zhanyun Tang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Functional Study ◽  
Histone Deacetylase 1 ◽  
Major Mechanism ◽  
Cellular Processes ◽  
Regulatory Enzymes ◽  
Substrate Proteins

Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a β-hydroxybutyrate–mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of β-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes.

Structural and functional similarities between the human cytoskeletal protein zyxin and the ActA protein of Listeria monocytogenes

1997 ◽  
Vol 110 (16) ◽  
pp. 1893-1906 ◽  
Author(s):  
R.M. Golsteyn ◽  
M.C. Beckerle ◽  
T. Koay ◽  
E. Friederich
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Cell Types ◽  
Striking Similarity ◽  
Rich Domain ◽  
Protein Partners ◽  
Localization Sequence

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells. Therefore we examined the possibility that an ActA-like protein is present in mammalian cells. By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types. We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box). Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences. A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells. Furthermore, zyxin and ActA exhibit common protein partners in vitro. On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells.

scholarly journals Investigation of Specific Substitutions in Virulence Genes Characterizing Phenotypic Groups of Low-Virulence Field Strains of Listeria monocytogenes

2005 ◽  
Vol 71 (10) ◽  
pp. 6039-6048 ◽  
Author(s):  
S. M. Roche ◽  
P. Gracieux ◽  
E. Milohanic ◽  
I. Albert ◽  
I. Virlogeux-Payant ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Phospholipase C ◽  
Single Amino Acid ◽  
Cell Infection ◽  
Gene Group ◽  
Group Iii ◽  
Group I ◽  
Group Ii ◽  
Phosphatidylinositol Phospholipase C

ABSTRACT Several models have shown that virulence varies from one strain of Listeria monocytogenes to another, but little is known about the cause of low virulence. Twenty-six field L. monocytogenes strains were shown to be of low virulence in a plaque-forming assay and in a subcutaneous inoculation test in mice. Using the results of cell infection assays and phospholipase activities, the low-virulence strains were assigned to one of four groups by cluster analysis and then virulence-related genes were sequenced. Group I included 11 strains that did not enter cells and had no phospholipase activity. These strains exhibited a mutated PrfA; eight strains had a single amino acid substitution, PrfAK220T, and the other three had a truncated PrfA, PrfAΔ174-237. These genetic modifications could explain the low virulence of group I strains, since mutated PrfA proteins were inactive. Group II and III strains entered cells but did not form plaques. Group II strains had low phosphatidylcholine phospholipase C activity, whereas group III strains had low phosphatidylinositol phospholipase C activity. Several substitutions were observed for five out of six group III strains in the plcA gene and for one out of three group II strains in the plcB gene. Group IV strains poorly colonized spleens of mice and were practically indistinguishable from fully virulent strains on the basis of the above-mentioned in vitro criteria. These results demonstrate a relationship between the phenotypic classification and the genotypic modifications for at least group I and III strains and suggest a common evolution of these strains within a group.

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