Arginine uptake and degradation systems are common throughout bacteria and archaea. The genome of human pathogen Chlamydia pneumoniae encodes three proteins now called AaxA, AaxB, and AaxC which function together to take up arginine, decarboxylate it, and expel the decarboxylation product, agmatine. AaxB is the previously characterized pyruvoyl-dependent arginine decarboxylase, AaxC is an inner membrane amino acid transport protein that functions as an arginine-agmatine antiporter, and AaxA is an outer membrane porin, which facilitates the uptake of arginine and also functions as a general porin with broad specificity. C. pneumoniae is a non-typical Gram negative bacteria and an obligate intracellular parasite with a unique 2-phase life cycle. The role of this system for arginine-agmatine exchange has yet to be determined but it may function to deplete host cell arginine as a means of inactivating host inducible nitric oxide synthase (iNOS), a molecule used in the innate immune response that has been shown to have an inhibitory affect on the growth of C. pneumoniae in cell culture. AaxB and AaxC are able to complement the loss of extreme acid-resistance in E. coli mutants that lack their own system for arginine-agmatine exchange, making pH homeostasis another possible role for this system. The porin AaxA is able to enhance arginine-agmatine exchange by AaxB and AaxC in E. coli mutants as well as by the native arginine decarboxylase AdiA and the native arginine-agmatine antiporter AdiC in wild type E. coli. AaxA is not an arginine-specific porin and instead acts as a general porin with a broad specificity. AaxA discriminates only against large and negatively charged solute molecules, and therefore it may have a broad role in the uptake of various biomolecules essential for chlamydial growth in addition to its role as part of a system for arginine-agmatine exchange. / text

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Dans le vin, les amines biogènes sont essentiellement d'origine microbienne, et sont produitesnotamment par les bactéries lactiques Oenococcus oeni, principal agent responsable de lafermentation malolactique, possède de nombreuses auxotrophies vis-à-vis des acides aminés.Aucune étude n'a été menée sur la relation entre l’auxotrophie vis-à-vis d’un acide aminé et leniveau de l’amine correspondante. 80 souches de Oenococcus oeni ont été isolées de vinsrouges. Leurs auxotrophies vis-à-vis d’acides aminés précurseurs d’amines biogènes (Arg,Tyr, His et Phe) ainsi que la présence de gènes codant des enzymes impliquées dans lasynthèse des amines ont été étudiées. Aucune relation entre auxotrophie et niveau deproduction d’amines ne peut être établie pour les souches testées. La présence de gènes codantdes enzymes impliquées dans la synthèse d’amines n’est pas non plus corrélée avec laproduction effective d’amines. Nous montrons pour la première fois que Oenococcus oeni estun producteur d’agmatine. Cette production est étroitement liée à la souche bactérienne. Lasouche la plus adaptée au milieu acide est celle qui consomme le plus l’arginine et enproportions équivalentes par les 2 voies : voie de l’arginine déiminase et voie de l’argininedécarboxylase. L’effet d’une addition d’agmatine dans des vins montre une atténuation del’effet boisé du Chardonnay sans doute par formation de base de Schiff ente les composésd’arôme et cette amine / In wine biogenic amines (BA) are mainly of microbial origin, Oenococcus oeni, the mainresponsible for malolactic fermentation, has been identified as a BA producer from nitrogenprecursors. Oenococcus oeni possess numerous amino acid auxotrophies that are precursors ofbiogenic amines. No study has been done so far to look at the relationship betweenauxotrophy for amino acids precursors of BA and the level of BA in the medium. In order todo so, 80 Oenococcus oeni strains were isolated from red wines. The detection of genesencoding the different decarboxylases responsible for BA synthesis has been realised. Inparallel, the auxotrophy for the four amino acids (Arg, Tyr, His, Phe) precursors of BA hascharacterized. Our results demonstrate that there is not direct correlation between auxotrophyand the accumulation of the corresponding BA as well as between the presence ofdecarboxylase gene and the accumulation of the corresponding BA. High levels of agmatineproduced from arginine decarboxylation by Oenococcus oeni is reported for the first time.Agmatine production is strain dependant. the most adapted to acidic environment is the strainwhith use arginine in higher level with same proportion for ADI pathway and argininedecarboxylase. Agmatine addition in wines reduce woody aroma probably by formation ofsciff bases between aromla compounds and amine

Agmatine, an endogenous amine derived from decarboxylation of l-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study, we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to 2 h immobilization stress daily for 7 days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with β-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50 mg/kg/day), i.p.). Likewise, endogenous agmatine levels measured by high-performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92 to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that the administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism.

The N-methyl-D-aspartate (NMDA) receptors are highly regulated ligand-gated ion channels, which are affected by many substrates. Overactivation of the NMDA receptor can lead to hyperexcitability and a number of neurotoxic effects and neurological diseases. Agmatine has been demonstrated to act allosterically as an inhibitory modulator at the polyamine recognition sites of the NMDA receptor complex. The present study synthesized and evaluated a library of agmatine analogs for their ability to displace tritiated MK-801 from NMDARs in P2 membrane preparations from rat brains at ligand concentrations of 1 mM and 50 uM. A full dose-response curve was generated for the most active compounds, in the presence and absence of a pathological level of spermidine (100 uM). A forty-five member subset of arylidenamino-guanidino compounds was synthesized and all were demonstrated to be NMDA receptor inhibitory modulators in the above assay. Three of these compounds generated biphasic curves, indicating activity at two binding sites: the postulated high-affinity agmatine binding site, and a low-affinity site (perhaps the channel itself). (4-Chlorobenzylidenamino)-guanidine hydrochloride demonstrated an IC50 of 3.6 uM at the former site and 124.5 uM at the latter. Several computer models were generated to direct further synthesis. Based on the structure-activity relationship of the arylidenamino-guanidino compounds, a pharmacophore model of the agmatine binding site of the NMDAR was proposed.

Agmatine is an endogenous amine derived from decarboxylation of arginine catalysed by arginine decarboxylase. Agmatine is considered a novel neuromodulator and possesses neuroprotective properties in the central nervous system. The present study examined whether agmatine has neuroprotective effects against repeated restraint stress-induced morphological changes in rat medial prefrontal cortex and hippocampus. Sprague-Dawley rats were subjected to 6 h of restraint stress daily for 21 days. Immunohistochemical staining with β-tubulin III showed that repeated restraint stress caused marked morphological alterations in the medial prefrontal cortex and hippocampus. Stress-induced alterations were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Interestingly, endogenous agmatine levels, as measured by high-performance liquid chromatography, in the prefrontal cortex and hippocampus as well as in the striatum and hypothalamus of repeated restraint rats were significantly reduced as compared with the controls. Reduced endogenous agmatine levels in repeated restraint animals were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. Moreover, administration of exogenous agmatine to restrained rats abolished increases of arginine decarboxylase protein levels. Taken together, these results demonstrate that exogenously administered agmatine has neuroprotective effects against repeated restraint-induced structural changes in the medial prefrontal cortex and hippocampus. These findings indicate that stress-induced reductions in endogenous agmatine levels in the rat brain may play a permissive role in neuronal pathology induced by repeated restraint stress.

In the present study, we examined the possible effect of chronic treatment with glucocorticoids on the morphology of the rat brain and levels of endogenous agmatine and arginine decarboxylase (ADC) protein, the enzyme essential for agmatine synthesis. Seven-day treatment with dexamethasone, at a dose (10 and 50 μg/kg/day) associated to stress effects contributed by glucocorticoids, did not result in obvious morphologic changes in the medial prefrontal cortex and hippocampus, as measured by immunocytochemical staining with β-tubulin III. However, 21-day treatment (50 μg/kg/day) produced noticeable structural changes such as the diminution and disarrangement of dendrites and neurons in these areas. Simultaneous treatment with agmatine (50 mg/kg/day) prevented these morphological changes. Further measurement with HPLC showed that endogenous agmatine levels in the prefrontal cortex and hippocampus were significantly increased after 7-day treatments with dexamethasone in a dose-dependent manner. On the contrary, 21-day treatment with glucocorticoids robustly reduced agmatine levels in these regions. The treatment-caused biphasic alterations of endogenous agmatine levels were also seen in the striatum and hypothalamus. Interestingly, treatment with glucocorticoids resulted in a similar change of ADC protein levels in most brain areas to endogenous agmatine levels: an increase after 7-day treatment versus a reduction after 21-day treatment. These results demonstrated that agmatine has neuroprotective effects against structural alterations caused by glucocorticoids in vivo. The parallel alterations in the endogenous agmatine levels and ADC expression in the brain after treatment with glucocorticoids indicate the possible regulatory effect of these stress hormones on the synthesis and metabolism of agmatine in vivo.

This dissertation is a compilation of published works and a manuscript that seek to understand the possible role of polyamines in the regulation of capsule in Streptococcus pneumoniae (Spn, pneumococcus). Spn remains a major health risk worldwide while the capsule is widely recognized as the principal virulence factor. Polyamines on the other hand are small hydrocarbon molecules known to regulate a number of cellular processes in bacteria. This work investigates the impact of deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine decarboxylase at the time of first and second publication), on protein expression and the capsule biosynthesis of virulent pneumococcal serotype 4 (TIGR4). We identify loss of capsular polysaccharide (CPS) in the deletion strain and based on proteomics results, we hypothesized that a shift in metabolism that favors the pentose phosphate pathway (PPP) over glycolytic pathway, that could reduce the availability of precursors for CPS had occurred. Comparison of transcriptomic and untargeted metabolomics profile of ∆SP_0916 with TIGR4 shows impaired glycolysis and Leloir pathways that provide CPS precursors, in the mutant strain. Furthermore, gene expression changes indicate possible reduction of common polyamines (cadaverine, putrescine, spermidine and spermine). Targeted metabolomics analysis confirmed reduced levels of polyamines in SP_0916. However, the result suggests that SP_0916 encodes an arginine decarboxylase, contrary to its existing annotation as a lysine decarboxylase in many bioinformatics databases. Biochemical characterization of the purified protein encoded by SP_0916 confirms that it is indeed catalyzes arginine decarboxylation, and exogenous supplementation of agmatine, the product of the reaction, successfully restores capsule biosynthesis. This study fixes an error in annotation of the TIGR4 genome and further establishes the essentiality of agmatine, a product of arginine decarboxylation as the key polyamine molecule modulating pneumococcal capsule. We later compared the impact of deletion of polyamine synthesis by gene deletion (ΔSP_0916) with chemical inhibition of synthesis using α- difluoromethylornithine (DFMO), in multiple pneumococcal serotypes. Results of this dissertation confirmed that pneumococcal pathways impacted by the disruption of polyamine biosynthesis either by gene deletion or chemical intervention are conserved and could regulate capsule synthesis.

Arginine decarboxylase is a key enzyme involved in the polyamine pathway of organisms. Pyruvoyl-dependent arginine decarboxylases are expressed in the form of proenzymes that self-cleave to form N-terminal [beta] and C-terminal [alpha] subunits generating an active pyruvoyl group at the [alpha] terminus. We have identified an archaeal homolog of a pyruvoyl-dependent arginine decarboxylase in Chlamydophila pneumoniae that could play a role in the persistence of the organism in the host. The recombinant enzyme showed highest activity at pH 3.4, which is the lowest optimum pH ever reported for a pyruvoyl dependent arginine decarboxylase. The proton-consuming decarboxylation raises intracellular pH, and thereby plays a role in acid-resistance. It could inhibit the pro-inflammatory nitric oxide synthase resulting in asymptomatic infection. A variant protein Thr⁵²Ser at the predicted cleavage site showed less pro-enzyme cleavage and activity compared to the wild-type. The homologs of arginine decarboxylase and flanking arginine-agmatine antiporter were also found in different biovariants of Chlamydia trachomatis. In the invasive L2 strain of C. trachomatis, the presence of a nonsense codon in the gene encoding arginine decarboxylase enzyme prevented the expression of an active enzyme. The variant protein with tryptophan replacing nonsense codon restored arginine decarboxylase activity. The non-invasive D strain of C. trachomatis had an intact arginine decarboxylase gene, but it was recombinantly expressed as a proenzyme that was uncleaved. The arginine-agmatine antiporters from both the strains were active and transported tritiated arginine into their cells. The polyamine pathway of the crenarchaeon Sulfolobus solfataricus uses arginine to make putrescine, but the organism lacks homologs of arginine decarboxylase. However, it has two paralogs of pyruvoyl dependent S-adenosylmethionine decarboxylase − SSO0536 and SSO0585. These enzymes were recombinantly expressed as pro-enzymes that self-cleaved into [beta] and [alpha] subunits. Even with a 47% amino acid sequence identity, the SSO0536 protein exhibited significant arginine decarboxylase activity whereas SSO0585 protein had significant S-adenosylmethionine decarboxylase activity. This is the first report of an S-adenosylmethionine decarboxylase enzyme showing alternative decarboxylase activity. The chimeric protein with the [alpha]-subunit of SSO0585 and [beta]-subunit of SSO0536 had arginine decarboxylase activity, suggesting that the residues responsible for substrate recognition are located in the amino terminus. / text