Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection

ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as se...

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Autores principales:	Chelsea E. Witte, Aaron T. Whiteley, Thomas P. Burke, John-Demian Sauer, Daniel A. Portnoy, Joshua J. Woodward
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/494b505188234fde880cd6ecca82a394
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spelling	oai:doaj.org-article:494b505188234fde880cd6ecca82a3942021-11-15T15:40:05ZCyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection10.1128/mBio.00282-132150-7511https://doaj.org/article/494b505188234fde880cd6ecca82a3942013-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00282-13https://doaj.org/toc/2150-7511ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. IMPORTANCE Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.Chelsea E. WitteAaron T. WhiteleyThomas P. BurkeJohn-Demian SauerDaniel A. PortnoyJoshua J. WoodwardAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 3 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Chelsea E. Witte Aaron T. Whiteley Thomas P. Burke John-Demian Sauer Daniel A. Portnoy Joshua J. Woodward Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection
description	ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. IMPORTANCE Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.
format	article
author	Chelsea E. Witte Aaron T. Whiteley Thomas P. Burke John-Demian Sauer Daniel A. Portnoy Joshua J. Woodward
author_facet	Chelsea E. Witte Aaron T. Whiteley Thomas P. Burke John-Demian Sauer Daniel A. Portnoy Joshua J. Woodward
author_sort	Chelsea E. Witte
title	Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection
title_short	Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection
title_full	Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection
title_fullStr	Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection
title_full_unstemmed	Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection
title_sort	cyclic di-amp is critical for <named-content content-type="genus-species">listeria monocytogenes</named-content> growth, cell wall homeostasis, and establishment of infection
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/494b505188234fde880cd6ecca82a394
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Cyclic di-AMP Is Critical for <named-content content-type="genus-species">Listeria monocytogenes</named-content> Growth, Cell Wall Homeostasis, and Establishment of Infection

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