Structural basis for c-di-AMP-dependent regulation of the bacterial stringent response by receptor protein DarB.

The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.

Association between contraceptive use and risk of lower urinary tract infection (LUTI): A case-control study.

AIM: The aim of this study was to analyze the association between widely used contraceptive methods and the manifestation of lower urinary tract infections (LUTI) in patients treated in gynecological practices in Germany. MATERIALS AND METHODS: This study was based on IQVIA Disease Analyzer database and includes a total of 133,638 females aged between 16 and 50 years who received an initial diagnosis of LUTI including cystitis (ICD-10: N39.0, N30.0) between January 2011 and December 2020 (index date). 1 : 1 matching of LUTI cases to non-LUTI controls was used to investigate the association between predefined criteria and LUTI. A greedy nearest neighbor propensity score method was used to balance cases and controls with respect to age, pregnancy, visit frequency during the observation period, and comorbidities including cancer, diabetes mellitus, and urolithiasis. Univariate logistic regression models were used to assess the association between contraceptive prescriptions and LUTI. RESULTS: The general use of any contraceptive method was negatively associated with subsequent LUTI. Injectable contraceptives and pills were negatively associated with LUTI manifestation. There was a significant negative association between monophasic preparations containing < 50 µg estrogen, triphasic preparations, and progestogen-only preparations and LUTI. By contrast, we found a significant positive association between emergency contraceptives and LUTI. CONCLUSION: The general application of birth control methods as well as the use of injectable contraceptives and oral contraceptives were negatively associated with LUTI manifestation. In contrast to other birth control methods, the intake of emergency contraception was positively associated with a manifestation of LUTI.

(Absence of) Association Between Non-Vitamin K Antagonist Oral Anticoagulant Therapy and Urinary Tract Infection in Patients With Atrial Fibrillation.

ABSTRACT: The aim of the present study is to identify a potential association of urinary tract infections (UTI) in a large population of patients receiving oral anticoagulation therapy treated in general practices in Germany. This study contains patients diagnosed with atrial fibrillation who received at least one prescription of either non-vitamin K antagonist oral anticoagulation (NOAC) or vitamin K antagonists (VKA) within January 2015 and December 2018. The incidence of UTI was examined cumulatively on the basis of Kaplan-Meier methods and was complemented by incidence rates measured in cases per 1000 patient years. Sex-stratified Cox regressions were conducted to examine possible associations in specific sex groups. The study comprised 26,934 patients receiving NOAC therapy and 8121 patients treated with VKA agents. Within a period of 5 years, slightly more NOAC than VKA users were diagnosed with UTI (20.3% vs. 19.3%), whereas the incidence rate was slightly higher in patients receiving NOAC therapy than in those under VKA treatment (50.8 cases vs. 50.5 cases in 1000 patient years). There was no significant association between direct oral anticoagulants versus vitamin K antagonists and infections of the urinary tract. Our study did not identify any significant association between therapy with direct oral anticoagulants versus vitamin K anticoagulants and UTI in patients diagnosed with atrial fibrillation in general practices in Germany. Because current findings regarding the risk of UTI in patients receiving oral anticoagulation therapy remain limited and contradictory, further investigations including a broad patient population are necessary to determine patients at risk for UTI and reconcile conflicting evidence.

Association between antiseizure medication use and risk of urinary tract infection: A case-control study.

AIM: The aim of this study was to analyze the association between antiseizure medication (ASM) and the risk of urinary tract infections (UTI) in patients with epilepsy treated in general practices in Germany. METHODS: This study includes a total of 2201 patients (mean age: 61.4) whose first documented UTI diagnosis occurred between January 2015 and December 2019 (index date) and who were prescribed at least one ASM in 1198 general practices in Germany within one year prior to the index date. Based on a case-control design, the association between predefined criteria and UTI was investigated by matching (1:1) controls without UTI to cases with UTI by sex, age, and codiagnoses. Logistic regression models were used to analyze the association between ASM use and UTI risk. RESULTS: In the first regression model, phenytoin (PHT), primidone, carbamazepine (CBZ), and valproate (VPA) were associated with an increased risk of UTI. In the second model, these associations were confirmed with effects per prescription for PHT, primidone, CBZ, and VPA use. Additionally, the effect per prescription was significant for oxcarbazepine (OXC), topiramate, and gabapentin. CONCLUSION: The study found that PHT, primidone, CBZ, and VPA in particular are associated with an increased risk of infections of the urinary tract. Oxcarbazepine, topiramate, and gabapentin are also associated with increased risk of UTI, albeit to a less significant extent. In general, the immunological and hematological side effects of these molecules may play an important role in the development of UTI under anticonvulsant therapy.

Crystal structures of the c-di-AMP-synthesizing enzyme CdaA.

Cyclic di-AMP (c-di-AMP) is the only second messenger known to be essential for bacterial growth. It has been found mainly in Gram-positive bacteria, including pathogenic bacteria like Listeria monocytogenes CdaA is the sole diadenylate cyclase in L. monocytogenes, making this enzyme an attractive target for the development of novel antibiotic compounds. Here we report crystal structures of CdaA from L. monocytogenes in the apo state, in the post-catalytic state with bound c-di-AMP and catalytic Co2+ ions, as well as in a complex with AMP. These structures reveal the flexibility of a tyrosine side chain involved in locking the adenine ring after ATP binding. The essential role of this tyrosine was confirmed by mutation to Ala, leading to drastic loss of enzymatic activity.

An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes.

The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c-di-AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram-positive pathogen Listeria monocytogenes relies on the membrane-bound diadenylate cyclase CdaA for c-di-AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c-di-AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c-di-AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c-di-AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.

Making and Breaking of an Essential Poison: the Cyclases and Phosphodiesterases That Produce and Degrade the Essential Second Messenger Cyclic di-AMP in Bacteria.

Cyclic di-AMP is a second-messenger nucleotide that is produced by many bacteria and some archaea. Recent work has shown that c-di-AMP is unique among the signaling nucleotides, as this molecule is in many bacteria both essential on one hand and toxic upon accumulation on the other. Moreover, in bacteria, like Bacillus subtilis, c-di-AMP controls a biological process, potassium homeostasis, by binding both potassium transporters and riboswitch molecules in the mRNAs that encode the potassium transporters. In addition to the control of potassium homeostasis, c-di-AMP has been implicated in many cellular activities, including DNA repair, cell wall homeostasis, osmotic adaptation, biofilm formation, central metabolism, and virulence. c-di-AMP is synthesized and degraded by diadenylate cyclases and phosphodiesterases, respectively. In the diadenylate cyclases, one type of catalytic domain, the diadenylate cyclase (DAC) domain, is coupled to various other domains that control the localization, the protein-protein interactions, and the regulation of the enzymes. The phosphodiesterases have a catalytic core that consists either of a DHH/DHHA1 or of an HD domain. Recent findings on the occurrence, domain organization, activity control, and structural features of diadenylate cyclases and phosphodiesterases are discussed in this review.

A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA.

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

Association between Antihypertensive Therapy and Risk of Acute Lower Respiratory Infections (ALRI): A Retrospective Cohort Study.

Purpose: The aim of this study was to analyze the association between antihypertensive drugs and the incidence of acute lower respiratory infections in patients treated in general practices in Germany. Methods: After propensity score matching of five antihypertensive drug classes, a total of 377,470 patients aged ≥18 years were available for analysis. The association between each antihypertensive drug class and ALRI incidence as compared to all other antihypertensive drug classes (as a group) was studied using conditional Cox regression analyses. Because of multiple comparisons and large patient samples, findings were clinically considered relevant when the hazard ratio was <0.85 or >1.15. Results: The regression analyses applied found no clinically relevant associations between antihypertensive drugs and the incidence of acute lower respiratory infections, as all hazard ratios were between 0.85 and 1.15. Conclusion: In the present study, only slight and not clinically relevant increases or decreases in the ALRI incidence were observed. Additional studies are necessary to further explore the risks associated with antihypertensive agents that are widely embedded in today's clinical practice.

A meet-up of two second messengers: the c-di-AMP receptor DarB controls (p)ppGpp synthesis in Bacillus subtilis.

Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB-Rel interaction results in the Rel-dependent accumulation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.