Antibiotic-resistant strains of Staphylococcus aureus are being viewed as a serious threat by various public health agencies. Identifying novel targets in this important pathogen is crucial to the development of new effective antibacterial formulations. We investigated the antibacterial effect of a colloidal nanosilver formulation, Silversol®, against an antibiotic-resistant strain of S. aureus using appropriate in vitro assays. Moreover, we deciphered the molecular mechanisms underlying this formulation\'s anti-S. aureus activity using whole transcriptome analysis. Lower concentrations of the test formulation exerted a bacteriostatic effect against this pathogen, and higher concentrations exerted a bactericidal effect. Silversol® at sub-lethal concentration was found to disturb multiple physiological traits of S. aureus such as growth, antibiotic susceptibility, membrane permeability, efflux, protein synthesis and export, biofilm and exopolysaccharide production, etc. Transcriptome data revealed that the genes coding for transcriptional regulators, efflux machinery, transferases, β-lactam resistance, oxidoreductases, metal homeostasis, virulence factors, and arginine biosynthesis are expressed differently under the influence of the test formulation. Genes (argG and argH) involved in arginine biosynthesis emerged among the major targets of Silversol®\'s antibacterial activity against S. aureus.

NADP/NADPH plays an indispensable role in cellular metabolism, serving as a pivotal cofactor in numerous enzymatic processes involved in anabolic pathways, antioxidant defense, and the biosynthesis of essential cellular components. NAD/NADH kinases (NADKs) phosphorylate NAD/NADH, constituting the sole de novo synthetic pathway for NADP/NADPH generation. Despite the pivotal role of NADP/NADPH in cellular functions, the physiological role of NADK remains largely unexplored in filamentous fungi. In this study, we identified three putative NADKs in Fusarium graminearum-FgNadk1, FgNadk2, and FgNadk3-responsible for NAD/NADH phosphorylation. NADK-mediated formation of intracellular NADPH proved crucial for vegetative growth, sexual reproduction, and virulence. Specifically, FgNadk2, the mitochondrial NADK, played a role in oxidative stress resistance and the maintenance of mitochondrial reactive oxygen species levels. Moreover, the deletion of FgNADK2 resulted in arginine auxotrophy, contributing to the reduced fungal virulence. These findings underscore the necessity of mitochondrial NADK in fungal virulence in F. graminearum, revealing its involvement in mitochondrial redox homeostasis and the arginine biosynthetic pathway. This study provides critical insights into the interconnectedness of metabolic pathways essential for fungal growth, stress response, and pathogenicity.

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.
CONCLUSIONS: This study revealed the catalytic efficiency and inhibition of cyanobacterial argininosuccinate synthetase by arginine and demonstrated that a strain overexpressing this enzyme grew faster than the wild-type strain.

Genome sequencing has demonstrated that Staphylococcus aureus encodes arginine biosynthetic genes argDCJBFGH synthesizing proteins that mediate arginine biosynthesis using glutamate as a substrate. Paradoxically, however, S. aureus does not grow in a defined, glutamate-replete medium lacking arginine and glucose (CDM-R). Studies from our laboratory have found that specific mutations are selected by S. aureus that facilitate growth in CDM-R. However, these selected mutants synthesize arginine utilizing proline as a substrate rather than glutamate. In this study, we demonstrate that the ectopic expression of the argDCJB operon supports the growth of S. aureus in CDM-R, thus documenting the functionality of this pathway. Furthermore, suppressor mutants of S. aureus JE2 putA::Tn, which is defective in synthesizing arginine from proline, were selected on CDM-R agar. Genome sequencing revealed that these mutants had compensatory mutations within both spoVG, encoding an ortholog of the Bacillus subtilis stage V sporulation protein, and sarA, encoding the staphylococcal accessory regulator. Transcriptional studies document that argD expression is significantly increased when JE2 spoVG sarA was grown in CDM-R. Lastly, we found that a mutation in ahrC was required to induce argD expression in JE2 spoVG sarA when grown in an arginine-replete medium (CDM), suggesting that AhrC also functions to repress argDCJB in an arginine-dependent manner. In conclusion, these data indicate that the argDCJB operon is functional when transcribed in vitro and that SNPs within potential putative regulatory proteins are required to alleviate the repression.IMPORTANCEAlthough Staphylococcus aureus has the capability to synthesize all 20 amino acids, it is phenotypically auxotrophic for several amino acids including arginine. This work identifies putative regulatory proteins, including SpoVG, SarA, and AhrC, that function to inhibit the arginine biosynthetic pathways using glutamate as a substrate. Understanding the ultimate mechanisms of why S. aureus is selected to repress arginine biosynthetic pathways even in the absence of arginine will add to the growing body of work assessing the interactions between metabolism and S. aureus pathogenesis.

Halo blight is a plant disease that leads to a significant decrease in the yield of common bean crops and kiwi fruits. The infection is caused by Pseudomonas syringae pathovars that produce phaseolotoxin, an antimetabolite which targets arginine metabolism, particularly by inhibition of ornithine transcarbamylase (OTC). OTC is responsible for production of citrulline from ornithine and carbamoyl phosphate. Here we present the first crystal structures of the plant OTC from Arabidopsis thaliana (AtOTC). Structural analysis of AtOTC complexed with ornithine and carbamoyl phosphate reveals that OTC undergoes a significant structural transition when ornithine enters the active site, from the opened to the closed state. In this study we discuss the mode of OTC inhibition by phaseolotoxin, which seems to be able to act only on the fully opened active site. Once the toxin is proteolytically cleaved, it mimics the reaction transition state analogue to fit inside the fully closed active site of OTC. Additionally, we indicate the differences around the gate loop region which rationally explain the resistance of some bacterial OTCs to phaseolotoxin.

UNASSIGNED: Liu-Wei-Luo-Bi (LWLB) granules was a Chinese compound prescription for treating diabetic peripheral neuropathy (DPN). The aim of this study was to investigate the effect of LWLB granules on diabetic mice with peripheral neuropathy and to elucidate the potential mechanism based on an untargeted metabolomics approach.
UNASSIGNED: One hundred forty db/db mice were randomly divided into seven groups: the Control group, DPN group, Mudan (MD) granules group, Epalrestat (Epa) group, and the LWLB low, medium, or high dose (LW-l, LW-m, or LW-h) group. After 12 weeks of treatment, body weight, blood glucose, mechanical pain threshold, motor conduction velocity (MCV), sensory conduction velocity (SCV), and Pathological Organization of the Sciatic and Caudal Nerves in mice were measured. Serum samples were collected for untargeted metabolomics analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and multivariate statistics. Disease-related pathways were screened out with function enrichment analyses of candidate biomarkers.
UNASSIGNED: LWLB granules can improve the peripheral neuropathy of type 2 diabetic mice with peripheral nerve conduction disorders, mainly through significantly improving the nerve conduction velocity (P < 0.05) and lowering the mechanical pain threshold (P < 0.05). A total of 43 metabolites were identified as potential biomarkers related to the therapeutic effect of LWLB granules. Fifty, 4, and 26; 23, 4, and 22; and 24, 1, and 16 biomarkers were discovered in the LW-l, LW-m, and LW-h groups at the 4th, 6th, and 12th weeks, respectively. Five, three, seven, five, and four metabolic pathways were found in MD, Epa, LW-l, LW-m, and LW-h groups, respectively. The arginine biosynthesis pathway is the overlapping pathway in LW-l, LW-m, and LW-h groups.
UNASSIGNED: LWLB granules have an obvious neuroprotective effect on diabetic peripheral neuropathy, and the metabolism mechanism of LWLB is mainly related to the arginine biosynthesis pathway on diabetic db/db mice with peripheral neuropathy.

Adherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn\'s disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers.
Comparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers.
Comparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection.
This is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification.

Multidrug-resistant gram-negative pathogens such as Escherichia coli have become increasingly difficult to treat and therefore alternative treatment options are needed. Targeting virulence factors like biofilm formation could be one such option. Inhibition of biofilm-related structures like curli and cellulose formation in E. coli has been shown for different phenolic natural compounds like epigallocatechin gallate. This study demonstrates this effect for other structurally unrelated phenolics, namely octyl gallate, scutellarein and wedelolactone. To verify whether these structurally different compounds influence identical pathways of biofilm formation in E. coli a broad comparative RNA-sequencing approach was chosen with additional RT-qPCR to gain initial insights into the pathways affected at the transcriptomic level. Bioinformatical analysis of the RNA-Seq data was performed using DESeq2, BioCyc and KEGG Mapper. The comparative bioinformatics analysis on the pathways revealed that, irrespective of their structure, all compounds mainly influenced similar biological processes. These pathways included bacterial motility, chemotaxis, biofilm formation as well as metabolic processes like arginine biosynthesis and tricarboxylic acid cycle. Overall, this work provides the first insights into the potential mechanisms of action of novel phenolic biofilm inhibitors and highlights the complex regulatory processes of biofilm formation in E. coli.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

To evaluate whether nicotinamide adenine dinucleotide-positive (NAD+) boosting modulates adaptive immunity, primary CD4+ T cells from healthy control and psoriasis subjects were exposed to vehicle or nicotinamide riboside (NR) supplementation. NR blunts interferon γ (IFNγ) and interleukin (IL)-17 secretion with greater effects on T helper (Th) 17 polarization. RNA sequencing (RNA-seq) analysis implicates NR blunting of sequestosome 1 (sqstm1/p62)-coupled oxidative stress. NR administration increases sqstm1 and reduces reactive oxygen species (ROS) levels. Furthermore, NR activates nuclear factor erythroid 2-related factor 2 (Nrf2), and genetic knockdown of nrf2 and the Nrf2-dependent gene, sqstm1, diminishes NR amelioratory effects. Metabolomics analysis identifies that NAD+ boosting increases arginine and fumarate biosynthesis, and genetic knockdown of argininosuccinate lyase ameliorates NR effects on IL-17 production. Hence NR via amino acid metabolites orchestrates Nrf2 activation, augments CD4+ T cell antioxidant defenses, and attenuates Th17 responsiveness. Oral NR supplementation in healthy volunteers similarly increases serum arginine, sqstm1, and antioxidant enzyme gene expression and blunts Th17 immune responsiveness, supporting evaluation of NAD+ boosting in CD4+ T cell-linked inflammation.