No abstract

bCeliac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of cereal gluten proteins. This disorder is associated with imbalances in the gut microbiota composition that could be involved in the pathogenesis of CD. The aim of this study was to characterize the composition and diversity of the cultivable duodenal mucosa-associated bacteria of CD patients and control children. Duodenal biopsy specimens from patients with active disease on a gluten-containing diet (n ‫؍‬ 32), patients with nonactive disease after adherence to a gluten-free diet (n ‫؍‬ 17), and controls (n ‫؍‬ 8) were homogenized and plated on plate count agar, Wilkins-Chalgren agar, brain heart agar, or yeast, Casitone, and fatty acid agar. The isolates were identified by partial 16S rRNA gene sequencing. Renyi diversity profiles showed the highest diversity values for active CD patients, followed by nonactive CD patients and control individuals. Members of the phylum Proteobacteria were more abundant in patients with active CD than in the other child groups, while those of the phylum Firmicutes were less abundant. Members of the families Enterobacteriaceae and Staphylococcaceae, particularly the species Klebsiella oxytoca, Staphylococcus epidermidis, and Staphylococcus pasteuri, were more abundant in patients with active disease than in controls. In contrast, members of the family Streptococcaceae were less abundant in patients with active CD than in controls. Furthermore, isolates of the Streptococcus anginosus and Streptococcus mutans groups were more abundant in controls than in both CD patient groups, regardless of inflammatory status. The findings indicated that the disease is associated with the overgrowth of possible pathobionts that exclude symbionts or commensals that are characteristic of the healthy small intestinal microbiota.

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of ϳ4, and the 50% inhibitory arginine concentration (I 0.5 ) was shown to increase with temperature, approaching above 65°C the I 0.50 observed at 37°C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75°C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the K m for acetylglutamate triggered by the inhibitor, but at 37°C arginine also substantially decreased the V max of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.Thermotoga maritima, one of the most highly thermophilic eubacteria (optimum growth temperature, 80°C) (20) and possibly one of the deepest branching and more slowly evolving of the eubacterial lineages (2), has been the subject of an already completed genome sequencing project (33) and, given its possible evolutionary position and biotechnological potential (19), is also the target of one of the few structural genomics projects being developed now (25).To exploit to its maximum the completeness of the genomic information and the massive structural data expected, it would be highly desirable to have a detailed knowledge of the physiology and metabolism of T. maritima rather than the patchy existing knowledge (3). Concerning the object of the present work, arginine biosynthesis, crude T. maritima extracts were shown to exhibit (36) enzyme activity

N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.

No abstract

N-acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification of NAGS gene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. Overall, 36 NAGS mutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele. 61% of mutations are missense changes. Lack or decrease of NAGS protein is predicted for ∼1/3 of mutations. Missense mutations frequency is inhomogeneous along NAGS: null for exon 1, but six in exon 6, which reflects the paramount substrate binding/catalytic role of the C-terminal domain (GNAT domain). Correspondingly, phenotypes associated with missense mutations mapping in the GNAT domain are more severe than phenotypes of amino acid kinase domain-mapping missense mutations. Enzyme activity and stability assays with 12 mutations introduced into pure recombinant Pseudomonas aeruginosa NAGS, together with in silico structural analysis, support the pathogenic role of most NAGSD-associated mutations found. The disease-causing mechanisms appear to be, from higher to lower frequency, decreased solubility/stability, aberrant kinetics/catalysis, and altered arginine modulation.

No abstract

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2- C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure-activity relationships by probing the ability of MMV008138 analogs to inhibit PfIspD recombinant enzyme. Here, we report PfIspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and PfIspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents.