The formation of the organized bacterial community called biofilm is a crucial event in bacterial physiology. Given that biofilms are often refractory to antibiotics and disinfectants to which ...planktonic bacteria are susceptible, their formation is also an industrially and medically relevant issue. Pseudomonas aeruginosa, a well-known human pathogen causing acute and chronic infections, is considered a model organism to study biofilms. A large number of environmental cues control biofilm dynamics in bacterial cells. In particular, the dispersal of individual cells from the biofilm requires metabolic and morphological reprogramming in which the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) plays a central role. The diatomic gas nitric oxide (NO), a well-known signaling molecule in both prokaryotes and eukaryotes, is able to induce the dispersal of P. aeruginosa and other bacterial biofilms by lowering c-di-GMP levels. In this review, we summarize the current knowledge on the molecular mechanisms connecting NO sensing to the activation of c-di-GMP-specific phosphodiesterases in P. aeruginosa, ultimately leading to c-di-GMP decrease and biofilm dispersal.
Melanoma is the most fatal form of skin cancer, with increasing prevalence worldwide. The most common melanoma genetic driver is mutation of the proto-oncogene serine/threonine kinase
BRAF
; thus, ...the inhibition of its MAP kinase pathway by specific inhibitors is a commonly applied therapy. However, many patients are resistant, or develop resistance to this type of monotherapy, and therefore combined therapies which target other signaling pathways through various molecular mechanisms are required. A possible strategy may involve targeting cellular energy metabolism, which has been recognized as crucial for cancer development and progression and which connects through glycolysis to cell surface glycan biosynthetic pathways. Protein glycosylation is a hallmark of more than 50% of the human proteome and it has been recognized that altered glycosylation occurs during the metastatic progression of melanoma cells which, in turn facilitates their migration. This review provides a description of recent advances in the search for factors able to remodel cell metabolism between glycolysis and oxidative phosphorylation, and of changes in specific markers and in the biophysical properties of cells during melanoma development from a nevus to metastasis. This development is accompanied by changes in the expression of surface glycans, with corresponding changes in ligand-receptor affinity, giving rise to structural features and viscoelastic parameters particularly well suited to study by label-free biophysical methods.
Serine hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of l-serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate. This enzyme, which plays a pivotal role in ...one-carbon metabolism, is involved in cancer metabolic reprogramming and is a recognized target of chemotherapy intervention. In humans, two isoforms of the enzyme exist, which are commonly termed cytosolic SHMT1 and mitochondrial SHMT2. Considerable attention has been paid to the structural, mechanistic, and metabolic features of these isozymes. On the other hand, a detailed comparison of their catalytic and regulatory properties is missing, although this aspect seems to be considerably important, considering that SHMT1 and SHMT2 reside in different cellular compartments, where they play distinct roles in folate metabolism. Here we performed a full kinetic characterization of the serine hydroxymethyltransferase reaction catalyzed by SHMT1 and SHMT2, with a focus on pH dependence and substrate inhibition. Our investigation, which allowed the determination of all kinetic parameters of serine hydroxymethyltransferase forward and backward reactions, uncovered a previously unobserved substrate inhibition by l-serine and highlighted several interesting differences between SHMT1 and SHMT2. In particular, SHMT2 maintains a pronounced tetrahydrofolate substrate inhibition even at the alkaline pH characteristic of the mitochondrial matrix, whereas with SHMT1 this is almost abolished. At this pH, SHMT2 also shows a catalytic efficiency that is much higher than that of SHMT1. These observations suggest that such different properties represent an adaptation of the isoforms to the respective cellular environments and that substrate inhibition may be a form of regulation.
Prostate cancer is one of the most common types of cancer in western country males but the mechanisms involved in the transformation processes have not been clearly elucidated. Alteration in cellular ...metabolism in cancer cells is recognized as a hallmark of malignant transformation, although it is becoming clear that the biological features of metabolic reprogramming not only differ in different cancers, but also among different cells in a type of cancer. Normal prostate epithelial cells have a peculiar and very inefficient energy metabolism as they use glucose to synthesize citrate that is secreted as part of the seminal liquid. During the transformation process, prostate cancer cells modify their energy metabolism from inefficient to highly efficient, often taking advantage of the interaction with other cell types in the tumor microenvironment that are corrupted to produce and secrete metabolic intermediates used by cancer cells in catabolic and anabolic processes. We recapitulate the metabolic transformations occurring in the prostate from the normal cell to the metastasis, highlighting the role of the microenvironment and summarizing what is known on the molecular mechanisms involved in the process.
The nitrogen cycle pathways are responsible for the circulation of inorganic and organic N-containing molecules in nature. Among these pathways, those involving amino acids, N-oxides and in ...particular nitric oxide (NO) play strategic roles in the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond their role in the N-cycle, amino acids and NO are also signalling molecules able to influence group behaviour in microorganisms and cell-cell communication in multicellular organisms, including humans. In this minireview, we summarise the role of these compounds in the homeostasis of the bacterial communities called biofilms, commonly found in environmental, industrial and medical settings. Biofilms are difficult to eradicate since they are highly resistant to antimicrobials and to the host immune system. We highlight the effect of amino acids such as glutamate, glutamine and arginine and of NO on the signalling pathways involved in the metabolism of 3',5'-cyclic diguanylic acid (c-di-GMP), a master regulator of motility, attachment and group behaviour in bacteria. The study of the metabolic routes involving these N-containing compounds represents an attractive topic to identify targets for biofilm control in both natural and medical settings.
•HD-GYPs belong to HD-hydrolases and control cyclic dinucleotides levels.•HD-GYPs are related to biofilm, virulence and control of motility.•HD-GYPs are heterogenous in the metal binding site in ...terms of number and nature.•Additional efforts are need to depict the molecular mechanism of HD-GYP function.
The control of the intracellular level of cyclic dinucleotides is a major strategy to transduce external signals into a cellular response, particularly in bacteria. The HD-GYP metalloproteins, a subgroup of the larger family of histidine-aspartate (HD) hydrolases, can catalyze the cleavage of the phosphodiester bond(s) of cyclic dinucleotides. The HD signature is involved in metal binding in the active site, whereas the GYP motif is likely involved in recognition and interaction with other partners. The most representative substrate of HD-GYPs is the second messenger cyclic-di-GMP (c-di-GMP), a global regulator of bacterial biofilm, motility, and virulence. Hydrolysis of c-di-GMP into the linear form pGpG or into the monomeric guanosine derivative (GMP) reprograms the cellular phenotype, usually promoting biofilm dispersion and virulence. Recent data indicate that members of HD-GYP group can also hydrolyze the bacterial cyclic diGMP-AMP (cGAMP) dinucleotide or act as possible sensors of pGpG.
The HD-GYP diversity is not limited to substrate recognition: the most striking trait is the extraordinary heterogeneity of the active sites characterized so far, showing different metals ions assisting catalysis, both in terms of their nature and number. In this review, we provide a critical overview of the structural, functional, and kinetic properties of the bacterial HD-GYP metalloproteins.
The capability to obtain essential nutrients in hostile environments is a critical skill for pathogens. Under zinc‐deficient conditions, Pseudomonas aeruginosa expresses a pool of metal homeostasis ...control systems that is complex compared with other Gram‐negative bacteria and has only been partially characterized. Here, the structure and zinc‐binding properties of the protein PA4063, the first component of the PA4063–PA4066 operon, are described. PA4063 has no homologs in other organisms and is characterized by the presence of two histidine‐rich sequences. ITC titration detected two zinc‐binding sites with micromolar affinity. Crystallographic characterization, performed both with and without zinc, revealed an α/β‐sandwich structure that can be classified as a noncanonical ferredoxin‐like fold since it differs in size and topology. The histidine‐rich stretches located at the N‐terminus and between β3 and β4 are disordered in the apo structure, but a few residues become structured in the presence of zinc, contributing to coordination in one of the two sites. The ability to bind two zinc ions at relatively low affinity, the absence of catalytic cavities and the presence of two histidine‐rich loops are properties and structural features which suggest that PA4063 might play a role as a periplasmic zinc chaperone or as a concentration sensor useful for optimizing the response of the pathogen to zinc deficiency.
The structural and zinc‐binding properties of PA4063, one of the periplasmic proteins expressed by Pseudomonas aeruginosa under zinc‐deficient conditions, are presented. PA4063 has a noncanonical ferredoxin‐like fold. Two zinc‐binding sites with micromolar affinity are exposed and are located on the same face of the structure. Two histidine‐rich loops, which are disordered in the apoprotein, contribute to zinc coordination in one of the sites. These findings strongly suggest a role for PA4063 in zinc trafficking, possibly acting as a metal chaperone or as a regulator of a transport system.
Biofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial ...biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD from Caulobacter crescentus are described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were tested in vitro for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration IC50 of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent in vitro inhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation.
Biofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identified in silico and characterized in vitro by using the well-characterized DGC enzyme PleD from C. crescentus as a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds.
Abstract
Amino acids are crucial in nitrogen cycling and to shape the metabolism of microorganisms. Among them, arginine is a versatile molecule able to sustain nitrogen, carbon, and even ATP supply ...and to regulate multicellular behaviors such as biofilm formation. Arginine modulates the intracellular levels of 3′–5′cyclic diguanylic acid (c-di-GMP), a second messenger that controls biofilm formation, maintenance and dispersion. In Pseudomonas putida, KT2440, a versatile microorganism with wide biotechnological applications, modulation of c-di-GMP levels by arginine requires the transcriptional regulator ArgR, but the connections between arginine metabolism and c-di-GMP are not fully characterized. It has been recently demonstrated that arginine can be perceived by the opportunistic human pathogen Pseudomonas aeruginosa through the transducer RmcA protein (Redox regulator of c-di-GMP), which can directly decrease c-di-GMP levels and possibly affect biofilm architecture. A RmcA homolog is present in P. putida, but its function and involvement in arginine perceiving or biofilm life cycle had not been studied. Here, we present a preliminary characterization of the RmcA-dependent response to arginine in P. putida in modulating biofilm formation, c-di-GMP levels, and energy metabolism. This work contributes to further understanding the molecular mechanisms linking biofilm homeostasis and environmental adaptation.
A new connection between the amino acid arginine and the modulation of the biofilm lifestyle of P. putida.
In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of ...chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a
Pseudomonas aeruginosa
DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an
Escherichia coli
strain expressing the
wspR
gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn’s disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an
E
.
coli purH
mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by
P
.
aeruginosa
; however, it affected production of extracellular structures in
E
.
coli
clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in
E
.
coli
through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn’s disease.
Celotno besedilo
Dostopno za:
CEKLJ, DOBA, EMUNI, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK
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