C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression in Listeria monocytogenes.

C-di-AMP is an essential second messenger in many bacteria but its levels must be regulated. Unregulated c-di-AMP accumulation attenuates the virulence of many bacterial pathogens, including those that do not require c-di-AMP for growth. However, the mechanisms by which c-di-AMP regulates bacterial pathogenesis remain poorly understood. In Listeria monocytogenes , a mutant lacking both c-di-AMP phosphodiesterases, denoted as the ΔPDE mutant, accumulates a high c-di-AMP level and is significantly attenuated in the mouse model of systemic infection. All key L. monocytogenes virulence genes are transcriptionally upregulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Our transcriptomic analysis revealed that the ΔPDE mutant is significantly impaired for the expression of virulence genes within the PrfA core regulon. Subsequent quantitative gene expression analyses validated this phenotype both at the basal level and upon PrfA activation by GSH. A constitutively active PrfA * variant, PrfA G145S, which mimics the GSH-bound conformation, restores virulence gene expression in ΔPDE but only partially rescues virulence defect. Through GSH quantification and uptake assays, we found that the ΔPDE strain is significantly depleted for GSH, and that c-di-AMP inhibits GSH uptake. Constitutive expression of gshF (encoding a GSH synthetase) does not restore GSH levels in the ΔPDE strain, suggesting that c-di-AMP inhibits GSH synthesis activity or promotes GSH catabolism. Taken together, our data reveals GSH metabolism as another pathway that is regulated by c-di-AMP. C-di-AMP accumulation depletes cytoplasmic GSH levels within L. monocytogenes that leads to impaired virulence program expression. IMPORTANCE: C-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy, and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the mechanisms by which c-di-AMP accumulation diminishes bacterial pathogenesis are poorly understood. Such understanding will reveal the molecular function of c-di-AMP, and inform therapeutic development strategies. Here, we identify GSH metabolism as a pathway regulated by c-di-AMP that is pertinent to L. monocytogenes replication in the host. Given the role of GSH as a virulence signal, nutrient, and antioxidant, GSH depletion impairs virulence program expression and likely diminishes host adaptation.

Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence.

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 against 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. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis 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 suggests 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. IMPORTANCE Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol. GlmR is important for resistance to some cell-wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell-wall metabolite, UDP-GlcNAc, and suppressors that increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc, and this enzymatic activity is conserved in both Bacillus subtilis and Staphylococcus aureus. These results highlight the importance of accessory cell wall metabolism enzymes in responding to cell-wall stress in a variety of Gram-positive bacteria.

NrnA Is a Linear Dinucleotide Phosphodiesterase with Limited Function in Cyclic Dinucleotide Metabolism in Listeria monocytogenes.

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 ΔnrnA mutant had a mammalian cell infection defect that was fully restored by Escherichia 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 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 for 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.

The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology.

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole-genome sequencing was performed on cdaA mutants, and single-nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium Borrelia burgdorferi Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.

Characterization of Listeria monocytogenes isolates from lactating dairy cows in a Wisconsin farm: Antibiotic resistance, mammalian cell infection, and effects on the fecal microbiota.

Listeria monocytogenes is an invasive foodborne pathogen that is ubiquitously present in the dairy farm environment. Although cattle are a reservoir of L. monocytogenes, most adult animals do not exhibit clinical symptoms, suggesting a homeostasis between this pathogen and the bovine gastrointestinal ecosystem. Nevertheless, substantial prevalence of L. monocytogenes fecal shedding by dairy cattle has been reported in many studies, posing threats of transmission within the herd and contamination of the human food supply. Accordingly, understanding the L. monocytogenes ecology within the bovine gastrointestinal tract is important to prevent clinical illness in the animal host, reduce transmission, and guide intervention strategies. In this study, we conducted a longitudinal sampling of fecal samples from 20 lactating dairy cows in one Wisconsin farm over a 29-d period and found a strikingly high incidence of L. monocytogenes shedding, in 90% of sampled animals. The L. monocytogenes isolates were genetically diverse, representing all common serotypes previously identified from cattle. Additionally, most tested isolates were resistant to ampicillin, and a few were also resistant to gentamicin or trimethoprim/sulfamethoxazole. Most isolates effectively infected human epithelial cells (Caco-2) and murine fibroblasts (L2), suggesting that they are all capable of causing systemic infection if the intestinal barrier is breached. Finally, we investigated the effects of L. monocytogenes colonization on the gastrointestinal tract microbiota by analyzing the fecal bacterial communities of some shedding and nonshedding cows. Whereas L. monocytogenes did not affect the α and ß diversity of tested animals, a subset of shedding cows exhibited different abundances of certain operational taxonomic units within the Bacteroidetes and Firmicutes phyla compared with nonshedding cows. Overall, our findings highlight the threat of antibiotic resistance among some L. monocytogenes isolates, emphasize the need for a strain-specific approach in listeriosis treatment, and suggest the potential negative influence of subclinical L. monocytogenes carriage on animal gut health.

c-di-AMP Accumulation Impairs Muropeptide Synthesis in Listeria monocytogenes.

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.