scholarly journals Listeria monocytogenes GlmR is an accessory uridyltransferase essential for cytosolic survival and virulence

2021 ◽  
Author(s):  
Daniel Pensinger ◽  
Kimberly V Gutierrez ◽  
Hans B Smith ◽  
William J.B. Vincent ◽  
David M Stevenson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Responses ◽  
Mutational Analysis ◽  
Teichoic Acid ◽  
Wall Stress ◽  
Host Cells ◽  
Wall Teichoic Acid ◽  
Cell Wall Stress

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions for these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell wall stress, cytosolic survival, inflammasome avoidance and ultimately virulence in vivo. A genetic suppressor screen revealed that blocking utilization of UDP-GlcNAc by a non-essential wall teichoic acid decoration pathway restored resistance to cell wall stress and partially restored virulence of ΔglmR mutants. In parallel, metabolomics revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggest that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell wall stress responses and virulence in vivo. Taken together these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes.

scholarly journals Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

2021 ◽  
Author(s):  
Eric. T. Sumrall ◽  
Stephan R. Schneider ◽  
Samy Boulos ◽  
Martin J. Loessner ◽  
Yang Shen
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Teichoic Acid ◽  
Host Cells ◽  
Phage Resistance ◽  
Wall Teichoic Acid ◽  
Gram Positive ◽  
Cellular Invasion ◽  
Phage Adsorption ◽  
Listeria Ivanovii

Listeria ivanovii ( Liv ) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes ( Lmo ). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii , suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus. Importance Listeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes , strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacterium's major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

scholarly journals PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

2021 ◽  
Vol 17 (10) ◽  
pp. e1009881
Author(s):  
Jessica L. Kelliher ◽  
Caroline M. Grunenwald ◽  
Rhiannon R. Abrahams ◽  
McKenzie E. Daanen ◽  
Cassandra I. Lew ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Responses ◽  
Pathogenic Bacteria ◽  
Critical Role ◽  
Wall Stress ◽  
Wild Type ◽  
Comprehensive Overview ◽  
Bacterial Physiology ◽  
Cell Wall Stress

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.

scholarly journals Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

2021 ◽  
Author(s):  
Eric. T. Sumrall ◽  
Stephan R. Schneider ◽  
Samy Boulos ◽  
Martin J. Loessner ◽  
Yang Shen
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Teichoic Acid ◽  
Host Cells ◽  
Phage Resistance ◽  
Wall Teichoic Acid ◽  
Gram Positive ◽  
Cellular Invasion ◽  
Phage Adsorption ◽  
Listeria Ivanovii

AbstractListeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus.ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacterium’s major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

scholarly journals PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

2021 ◽  
Author(s):  
Jessica L. Kelliher ◽  
Caroline M. Grunenwald ◽  
Rhiannon R. Abrahams ◽  
McKenzie E. Daanen ◽  
Cassandra I. Lew ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Responses ◽  
Pathogenic Bacteria ◽  
Critical Role ◽  
Wall Stress ◽  
Wild Type ◽  
Comprehensive Overview ◽  
Bacterial Physiology ◽  
Cell Wall Stress

Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.

scholarly journals Hph1p and Hph2p, Novel Components of Calcineurin-Mediated Stress Responses in Saccharomyces cerevisiae

2004 ◽  
Vol 3 (3) ◽  
pp. 695-704 ◽  
Author(s):  
Victoria L Heath ◽  
Sidney L. Shaw ◽  
Sharmili Roy ◽  
Martha S. Cyert
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Stress Responses ◽  
Wall Stress ◽  
Sequence Motif ◽  
Alkaline Ph ◽  
Cell Wall Stress ◽  
High Concentrations

ABSTRACT Calcineurin is a Ca2+- and calmodulin-dependent protein phosphatase that plays a key role in animal and yeast physiology. In the yeast Saccharomyces cerevisiae, calcineurin is required for survival during several environmental stresses, including high concentrations of Na+, Li+, and Mn2+ ions and alkaline pH. One role of calcineurin under these conditions is to activate gene expression through its regulation of the Crz1p transcription factor. We have identified Hph1p as a novel substrate of calcineurin. HPH1 (YOR324C) and its homolog HPH2 (YAL028W) encode tail-anchored integral membrane proteins that interact with each other in the yeast two-hybrid assay and colocalize to the endoplasmic reticulum. Hph1p and Hph2p serve redundant roles in promoting growth under conditions of high salinity, alkaline pH, and cell wall stress. Calcineurin modifies the distribution of Hph1p within the endoplasmic reticulum and is required for full Hph1p activity in vivo. Furthermore, calcineurin directly dephosphorylates Hph1p and interacts with it through a sequence motif in Hph1p, PVIAVN. This motif is related to calcineurin docking sites in other substrates, such as NFAT and Crz1p, and is required for regulation of Hph1p by calcineurin. In contrast, Hph2p neither interacts with nor is dephosphorylated by calcineurin. Ca2+-induced Crz1p-mediated transcription is unaffected in hph1Δ hph2Δ mutants, and genetic analyses indicate that HPH1/HPH2 and CRZ1 act in distinct pathways downstream of calcineurin. Thus, Hph1p and Hph2p are components of a novel Ca2+- and calcineurin-regulated response required to promote growth under conditions of high Na+, alkaline pH, and cell wall stress.

scholarly journals Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response

2012 ◽  
Vol 333 (2) ◽  
pp. 109-120 ◽  
Author(s):  
Vanina Dengler ◽  
Patricia Stutzmann Meier ◽  
Ronald Heusser ◽  
Peter Kupferschmied ◽  
Judit Fazekas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Stress Response ◽  
Teichoic Acid ◽  
Wall Stress ◽  
Wall Teichoic Acid ◽  
Cell Wall Stress

scholarly journals An Antibiotic That Inhibits a Late Step in Wall Teichoic Acid Biosynthesis Induces the Cell Wall Stress Stimulon in Staphylococcus aureus

2012 ◽  
Vol 56 (4) ◽  
pp. 1810-1820 ◽  
Author(s):  
Jennifer Campbell ◽  
Atul K. Singh ◽  
Jonathan G. Swoboda ◽  
Michael S. Gilmore ◽  
Brian J. Wilkinson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Osmotic Stress ◽  
Late Stage ◽  
Teichoic Acid ◽  
Wall Stress ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Cell Wall Stress

ABSTRACTWall teichoic acids (WTAs) are phosphate-rich, sugar-based polymers attached to the cell walls of most Gram-positive bacteria. InStaphylococcus aureus, these anionic polymers regulate cell division, protect cells from osmotic stress, mediate host colonization, and mask enzymatically susceptible peptidoglycan bonds. Although WTAs are not required for survivalin vitro, blocking the pathway at a late stage of synthesis is lethal. We recently discovered a novel antibiotic, targocil, that inhibits a late acting step in the WTA pathway. Its target is TarG, the transmembrane component of the ABC transporter (TarGH) that exports WTAs to the cell surface. We examined here the effects of targocil onS. aureususing transmission electron microscopy and gene expression profiling. We report that targocil treatment leads to multicellular clusters containing swollen cells displaying evidence of osmotic stress, strongly induces the cell wall stress stimulon, and reduces the expression of key virulence genes, includingdltABCDand capsule genes. We conclude that WTA inhibitors that act at a late stage of the biosynthetic pathway may be useful as antibiotics, and we present evidence that they could be particularly useful in combination with beta-lactams.

scholarly journals Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

2021 ◽  
Author(s):  
Eric. T. Sumrall ◽  
Stephan R. Schneider ◽  
Samy Boulos ◽  
Martin J. Loessner ◽  
Yang Shen
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Teichoic Acid ◽  
Host Cells ◽  
Phage Resistance ◽  
Wall Teichoic Acid ◽  
Gram Positive ◽  
Cellular Invasion ◽  
Phage Adsorption ◽  
Listeria Ivanovii

AbstractListeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus.ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacterium’s major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

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