Presence of the arginine dihydrolase pathway in Mycoplasma

Barile, Michael F. (Division of Biologics Standards, National Institutes of Health, Bethesda, Md.), Robert T. Schimke, and Donald B. Riggs. Presence of the arginine dihydrolase pathway in Mycoplasma. J. Bacteriol. 91:189-192. 1966.-The presence of the arginine dihydrolase pathway was examined in 61 Mycoplasma strains representing at least 18 Mycoplasma species isolated from nine different sources: human, bovine, avian, murine, swine, goat, canine, sewage, and tissue cell culture origin. Some species were represented by only one or two strains. Different strains of the same species gave the same results. Ten species (56%) were positive. Many nonpathogenic Mycoplasma species (M. hominis, type 1 and 2, M. fermentans, M. salivarium, and M. gallinarum) were positive, whereas most pathogenic species (M. pneumoniae, M. gallisepticum, M. neurolyticum, and M. hyorhinis) were negative. The presence of arginine dihydrolase activity among Mycoplasma species may prove to be useful for purposes of identification and classification.     

Synthesis of the arginine dihydrolase pathway enzymes inLactobacillus buchneri

The formation of the arginine dihydrolase pathway enzymes inLactobacillus buchneri NCDO₁₁₀, a heterofermentative organism, was investigated. The specific activities of arginine deiminase, ornithine transcarbamylase, and carbamate kinase were higher in galactose-grown cells than in glucose- or sucrose-grown cells in the early stationary phase of growth. The addition of arginine to growing cells increased the specific activity of these three enzymes with all growth sugars. The specific activities of the enzymes decreased during the stationary phase of growth when the sugar-grown cells was galactose. When glucose was virtually exhausted from the medium, the activities of the three enzymes were not altered. This enzymic system was not repressed by glucose, and these results are different from those obtained withL. leichmanni, homofermentative organism.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, 6 September 1986.Member of the Scientific Researcher's Career of theConsejo Nacional de Investigaciones Cientificas Ténicas (CONICET) Argentina.     

Nitrosative stress induced cytotoxicity in Giardia intestinalis

AIMS: To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. METHODS AND RESULTS: Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. CONCLUSIONS: These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms.     

Arginine dihydrolase pathway in Lactobacillus buchneri: a review

The arginine dihydrolase system was studied in homo- and hetero-fermentative lactic acid bacteria. This system is widely distributed in Betabacteria lactobacilli subgroup (group II in Bergey's Manual). It is generally absent in the Thermobacterium lactobacilli subgroup (group IA in Bergey's Manual) and also in the Streptobacterium subgroup (group IB in Bergey's Manual). It is present in some species of the genus Streptococcus (groups II, III and IV in Bergey's Manual). In Lactobacillus buchneri NCDO110 the 3 enzymes of the arginine dihydrolase pathway, arginine deiminase, ornithine transcarbamylase and carbamate kinase, were purified and characterized. Arginine deiminase was partially purified (68-fold); ornithine transcarbamylase was also partially purified (14-fold), while carbamate kinase was purified to homogeneity. The apparent molecular weight of the enzymes was 199,000, 162,000 and 97,000 for arginine deiminase, ornithine transcarbamylase and carbamate kinase respectively. For arginine deiminase, maximum enzymatic activity was observed at 50 degrees C and pH 6; for ornithine transcarbamylase it was observed at 35 degrees C and pH 8.5, and for carbamate kinase at 30 degrees C and pH 5.4. The activation energy of the reactions was determined. For arginine deiminase, delta G* values were: 8,700 cal mol-1 below 50 degrees C and 380 cal mol-1 above 50 degrees C; for ornithine transcarbamylase, the values were: 9,100 cal mol-1 below 35 degrees C and 4,300 cal mol-1 above 35 degrees C; for carbamate kinase, the activation energy was: 4,078 cal mol-1 for the reaction with Mn2+ and 3,059 cal mol-1 for the reaction with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of Giardia intestinalis cyclophilin

The cyclophilins (Cyps) are family members of proteins that exhibit peptidylprolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosprin A (CsA) in varying degrees. During the process of random sequencing of a cDNA library made from Giardia intestinalis WB strain, the cyclophilin gene (gicyp 1) was isolated. An open reading frame of gicyp 1 gene was 576 nucleotides, which corresponded to a translation product of 176 amino acids (Gicyp 1). The identity with other Cyps was about 58-71%. The 13 residues that constituted the CsA binding site of human cyclophilin were also detected in the amino acid sequence of Gicyp 1, including tryptophan residue essential for the drug binding. The single copy of the gicyp 1 gene was detected in the G. intestinalis chromosome by southern hybridization analysis. Recombinant Gicyp 1 protein clearly accelerated the rate of cis-->trans isomerization of the peptide substrate and the catalysis was completely inhibited by the addition of 0.5 microM CsA.     

The superoxide reductase from the early diverging eukaryote Giardia intestinalis

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O₂^•−) not through its dismutation, but via reduction to hydrogen peroxide (H₂O₂) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR_Gi) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T_final) with Fe³⁺ ligated to glutamate or hydroxide depending on pH (apparent pK_a = 8.7). Although showing negligible SOD activity, reduced SOR_Gi reacts with O₂^•− with a pH-independent second-order rate constant k₁ = 1.0 × 10⁹ M^− 1 s^− 1 and yields the ferric-(hydro)peroxo intermediate T₁; this in turn rapidly decays to the T_final state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR_Gi is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.     

The O2-scavenging flavodiiron protein in the human parasite Giardia intestinalis

The flavodiiron proteins (FDP) are widespread among strict or facultative anaerobic prokaryotes, where they are involved in the response to nitrosative and/or oxidative stress. Unexpectedly, FDPs were fairly recently identified in a restricted group of microaerobic protozoa, including Giardia intestinalis, the causative agent of the human infectious disease giardiasis. The FDP from Giardia was expressed, purified, and extensively characterized by x-ray crystallography, stopped-flow spectroscopy, respirometry, and NO amperometry. Contrary to flavorubredoxin, the FDP from Escherichia coli, the enzyme from Giardia has high O(2)-reductase activity (>40 s(-1)), but very low NO-reductase activity (approximately 0.2 s(-1)); O(2) reacts with the reduced protein quite rapidly (milliseconds) and with high affinity (K(m) < or = 2 microM), producing H(2)O. The three-dimensional structure of the oxidized protein determined at 1.9A resolution shows remarkable similarities with prokaryotic FDPs. Consistent with HPLC analysis, the enzyme is a dimer of dimers with FMN and the non-heme di-iron site topologically close at the monomer-monomer interface. Unlike the FDP from Desulfovibrio gigas, the residue His-90 is a ligand of the di-iron site, in contrast with the proposal that ligation of this histidine is crucial for a preferential specificity for NO. We propose that in G. intestinalis the primary function of FDP is to efficiently scavenge O(2), allowing this microaerobic parasite to survive in the human small intestine, thus promoting its pathogenicity.     

Rapid tagging and integration of genes in Giardia intestinalis

We developed a series of plasmids that allow C-terminal tagging of any gene in its endogenous locus in Giardia intestinalis, with different epitope tags (triple hemagglutinin [3HA] and triple Myc [3Myc]) and selection markers (puromycin, neomycin, and a newly developed marker, blasticidin). Using these vectors, cyclin B and aurora kinase were tagged, expressed, and localized.     

Zoonotic genotype of Giardia intestinalis detected in a ferret

Giardia intestinalis has been found in a variety of mammals, including humans, and consists of host-specific and zoonotic genotypes. There has been only 1 study of G. intestinalis infection in weasels, but the genotype of its isolate remains unclear. In this study, we report the isolation of Giardia in a ferret exhibited at a pet shop. The isolate was analyzed genetically to validate the possibility of zoonotic transmission. Giardia diagnostic fragments of the small subunit ribosomal RNA, beta-giardin, and glutamate dehydrogenase genes were amplified from the ferret isolate and sequenced to reveal the phylogenetic relationships between it and other Giardia species or genotypes of G. intestinalis reported previously. The results showed that the ferret isolate represented the genetic group A-I in assemblage A, which could be a causative agent of human giardiasis.     

Characterization of the N-linked glycans of Giardia intestinalis

This article reports the first rigorous evidence for the existence of N-glycans in Giardia intestinalis, a parasite that is a widespread human pathogen, being a major cause of enteric disease in the world. Excreted/secreted molecules of G. intestinalis are known to stimulate the immune system. Structural strategies based on MALDI and electrospray mass spectrometry were employed to examine the excreted/secreted molecules for their N-glycan content. These revealed that the major oligosaccharides released by peptide N-glycosidase F are complex-type structures and correspond to bi-, and triantennary structures without core (alpha1,6) fucosylation. The major nonreducing epitopes in these complex-type glycans are: Galbeta1-4GlcNAc (LacNAc) and NeuAc alpha2-6Galbeta1-4GlcNAc (sialylated LacNAc).