The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles the regulation virulence-related factors pathogenic bacteria. A set P. genes, encoding proteins that contain putative domains characteristic diguanylate cyclases (DGCs) phosphodiesterases (PDEs) maintenance cellular levels second messenger...

Bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria activate either virulence pathways or biofilm formation. The mechanism c-di-GMP signal transduction, including recognition and subsequent phenotypic regulation, remain largely uncharacterized. key components these are various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition PilZ...

Summary The ubiquitous bacterial second messenger c‐di‐GMP regulates the expression of various virulence determinants in a wide range pathogens. Several studies have suggested that proteins with PilZ domain function as receptors. We identified Pseudomonas aeruginosa genome eight genes encoding for orhologues and demonstrated binding to all but one these direct ligand assay. One protein domain, Alg44, is involved biosynthesis extracellular polysaccharide alginate. shown increasing levels by...

Nucleotide signaling molecules are important messengers in key pathways that allow cellular responses to changing environments. Canonical secondary act through specific receptor proteins by direct binding alter their activity. Cyclic diadenosine monophosphate (c-di-AMP) is an essential molecule bacteria has only recently been discovered. Here we report on the identification of four Staphylococcus aureus c-di-AMP also widely distributed among other bacteria. Using affinity pull-down assay...

Pseudomonas aeruginosa utilizes a number of distinct pathways to secrete proteins that play various roles during infection. These include the type II secretion system, which is responsible for majority exoproducts into surrounding environment, including toxins and degradative enzymes. In contrast, III system mediates delivery protein effectors directly cytoplasm host cell. Using tissue culture assays mouse acute-pneumonia model, we have determined contribution each strain PAK, ExoS major...

Interactions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants metabolism, gene regulation, cellular homeostasis. Pharmaceuticals often target these interactions to interfere regulatory pathways. We have developed a rapid, precise, high-throughput method for quantitatively measuring protein-ligand without the need purify protein when performed in cells low background activity. This method, differential radial...

Pseudomonas aeruginosa is an opportunistic human pathogen that especially adept at forming surface-associated biofilms. P. causes catheter-associated urinary tract infections (CAUTIs) through biofilm formation on the surface of indwelling catheters. encodes three extracellular polysaccharides, PEL, PSL, and alginate, utilizes PEL PSL polysaccharides to form biofilms in vitro; however, requirement these during vivo not well understood. Here we show a murine model CAUTI PAO1, strain harboring...

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates variety of complex processes through diverse set c-di-GMP receptor proteins. We have utilized systematic approach to identify receptors from the pathogen Vibrio cholerae using Differential Radial Capillary Action Ligand Assay (DRaCALA). The DRaCALA screen identified majority known binding proteins in V. and revealed novel protein, MshE (VC0405), an ATPase associated with mannose sensitive hemagglutinin...

Significance Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that regulates complex behaviors such as biofilm formation. These are changed by altering the intracellular concentration of c-di-GMP. Degradation c-di-GMP occurs two-step process in which one set phosphodiesterases (PDE-As) linearize molecule into 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), followed hydrolysis unidentified (PDE-Bs) two GMPs. High levels pGpG inhibit PDE-As, and thus PDE-B activity important...

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa an important opportunistic pathogen utilizes cdiGMP-regulated polysaccharides, including alginate pellicle polysaccharide (PEL), mediate antibiotic resistance....

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttle-mediated metabolism support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells depth. In pathogenic bacterium Pseudomonas aeruginosa PA14, antibiotics called phenazines act oxidants balance...

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE proceeds as a direct translocation polypeptide between cells or in two distinct steps, each requiring specific signals for secretion across bacterial envelope Here, we used selective solubilization plasma membrane with digitonin measure during Yersinia infections HeLa cells. YopE, YopH, YopM YopN were found cytosol but not extracellular...

Pseudomonas aeruginosa is an opportunistic human pathogen that a key factor in the mortality of cystic fibrosis patients, and infection represents increased threat for health worldwide. Because resistance to antibiotics increasing, new inhibitors pharmacologically validated targets this bacterium are needed. Here we demonstrate cell-based yeast phenotypic assay, combined with large-scale inhibitor screen, identified small molecule can suppress toxicity caused by heterologous expression...

Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic heat resistance. In Lactococcus lactis, lactic acid bacterium which used the dairy industry as factory biotechnological only reported interaction partners of c-di-AMP are pyruvate carboxylase BusR, transcription regulator busAB operon for glycine betaine uptake. However, recent studies uncovered major role control potassium signal that...

Abstract The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses nutritional other stresses. There is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence pGpp in cells, perform comprehensive screening identify proteins that interact respectively pGpp, ppGpp pppGpp Bacillus species. Both involved...

Recently, there has been an explosion of research activities in the cyclic dinucleotides field. Cyclic dinucleotides, such as c-di-GMP and c-di-AMP, have shown to regulate bacterial virulence biofilm formation. can exist different aggregate forms, it demonstrated that polymorphism is influenced by nature cation present solution. In previous work, could only be at hundreds micromolar concentrations dinucleotide, a matter debate if exists under vivo conditions. this Article, we demonstrate...

In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. some c-di-AMP also binds the pyruvate carboxylase inhibit its activity. We have discovered in B. target protein DarB, rather than itself, specifically both vivo vitro. This interaction stimulates activity enzyme, as demonstrated vitro enzyme assays metabolite determinations. Both activation require apo-DarB are...

Abstract Dinucleases of the DEDD superfamily, such as oligoribonuclease, Rexo2 and nanoRNase C, catalyze essential final step RNA degradation, conversion di- to mononucleotides. The active sites these enzymes are optimized for substrates that two nucleotides long, do not discriminate between DNA. Here, we identified a novel subfamily, members which function dedicated deoxydinucleases (diDNases) specifically hydrolyze single-stranded DNA dinucleotides in sequence-independent manner. Crystal...

Successful establishment of Yersinia infections requires the type III machinery, a protein transporter that injects virulence factors (Yops) into macrophages. It is reported here pathway responds to environmental signals by transporting proteins distinct locations. enterocolitica cells sense an increase in extracellular amino acids (glutamate, glutamine, aspartate, and asparagine) results activation pathway. Another signal, provided serum such as albumin, triggers secretion YopD medium. The...

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