Dataset Information

Listeria monocytogenes strain-specific impairment of the TetR regulator underlies the drastic increase in cyclic di-AMP secretion and beta interferon-inducing ability.

ABSTRACT: Among a number of laboratory strains of Listeria monocytogenes used in experimental infection, strain LO28 is highly capable of inducing robust beta interferon (IFN-?) production in infected macrophages. In this study, we investigated the molecular mechanism of the IFN-?-inducing ability of LO28 by comparing it with that of strain EGD, a low-IFN-?-inducing strain. It was found that LO28 secretes a large amount of IFN-?-inducing factor, which turned out to be cyclic di-AMP. The secretion of cyclic di-AMP was dependent on MdrT, a multidrug resistance transporter, and LO28 exhibited a very high level of mdrT expression. The introduction of a null mutation into mdrT abolished the ability of LO28 to induce IFN-? production. Examination of genes responsible for the regulation of mdrT expression revealed a spontaneous 188-bp deletion in tetR of LO28. By constructing recombinant strains of LO28 and EGD in which tetR from each strain was replaced, it was confirmed that the distinct ability of LO28 is attributable mostly to tetR mutation. We concluded that the strong IFN-?-inducing ability of LO28 is due to a genetic defect in tetR resulting in the overexpression of mdrT and a concomitant increase in the secretion of cyclic di-AMP through MdrT.

SUBMITTER: Yamamoto T

PROVIDER: S-EPMC3416470 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Listeria monocytogenes strain-specific impairment of the TetR regulator underlies the drastic increase in cyclic di-AMP secretion and beta interferon-inducing ability.

Yamamoto Takeshi T Hara Hideki H Tsuchiya Kohsuke K Sakai Shunsuke S Fang Rendong R Matsuura Motohiro M Nomura Takamasa T Sato Fumihiko F Mitsuyama Masao M Kawamura Ikuo I

Infection and immunity 20120416 7

Among a number of laboratory strains of Listeria monocytogenes used in experimental infection, strain LO28 is highly capable of inducing robust beta interferon (IFN-β) production in infected macrophages. In this study, we investigated the molecular mechanism of the IFN-β-inducing ability of LO28 by comparing it with that of strain EGD, a low-IFN-β-inducing strain. It was found that LO28 secretes a large amount of IFN-β-inducing factor, which turned out to be cyclic di-AMP. The secretion of cycli ...[more]

PMID: 22508860

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Project description:Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger among bacteria. c-di-AMP regulates many cellular pathways through direct binding to several molecular targets in bacterial cells. c-di-AMP depletion is well known to destabilize the bacterial cell wall, resulting in increased bacteriolysis and enhanced susceptibility to cell wall targeting antibiotics. Using the human pathogen Listeria monocytogenes as a model, we found that c-di-AMP accumulation also impaired cell envelope integrity. An L. monocytogenes mutant deleted for c-di-AMP phosphodiesterases (pdeA pgpH mutant) exhibited a 4-fold increase in c-di-AMP levels and several cell wall defects. For instance, the pdeA pgpH mutant was defective for the synthesis of peptidoglycan muropeptides and was susceptible to cell wall-targeting antimicrobials. Among different muropeptide precursors, we found that the pdeA pgpH strain was particularly impaired in the synthesis of d-Ala-d-Ala, which is required to complete the pentapeptide stem associated with UDP-N-acetylmuramic acid (MurNAc). This was consistent with an increased sensitivity to d-cycloserine, which inhibits the d-alanine branch of peptidoglycan synthesis. Finally, upon examining d-Ala:d-Ala ligase (Ddl), which catalyzes the conversion of d-Ala to d-Ala-d-Ala, we found that its activity was activated by K+ Based on previous reports that c-di-AMP inhibits K+ uptake, we propose that c-di-AMP accumulation impairs peptidoglycan synthesis, partially through the deprivation of cytoplasmic K+ levels, which are required for cell wall-synthetic enzymes.IMPORTANCE The bacterial second messenger c-di-AMP is produced by a large number of bacteria and conditionally essential to many species. Conversely, c-di-AMP accumulation is also toxic to bacterial physiology and pathogenesis, but its mechanisms are largely undefined. We found that in Listeria monocytogenes, elevated c-di-AMP levels diminished muropeptide synthesis and increased susceptibility to cell wall-targeting antimicrobials. Cell wall defects might be an important mechanism for attenuated virulence in bacteria with high c-di-AMP levels.

Project description:The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenesIMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

Dataset Information

Listeria monocytogenes strain-specific impairment of the TetR regulator underlies the drastic increase in cyclic di-AMP secretion and beta interferon-inducing ability.

Publications

Listeria monocytogenes strain-specific impairment of the TetR regulator underlies the drastic increase in cyclic di-AMP secretion and beta interferon-inducing ability.

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