Eugenia Pennacchietti

Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon

For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH < 2.5) encountered during transit through the host stomach. The glutamate‐dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP‐dependent enzyme which performs a proton‐consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamatein/γ‐aminobutyrateout (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH‐dependent intracellular activation of E. coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non‐pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co‐regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA‐enriched foods possess health‐promoting properties.

Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB

Escherichia coli and other enterobacteria exploit the H+‐consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10‐fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N‐terminal 14 residues, involved in the formation of two triple‐helix bundles, were deleted by mutagenesis. X‐ray structures, obtained in the presence of the substrate analogue acetate, identified halide‐binding sites at the base of each N‐terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5′‐phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl− exchange activities of ClC‐ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH.

Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5′-phosphate-dependent enzyme catalyzing CO2 release from the α-carboxyl group of l-glutamate to yield γ-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the C termini and that the 340 nm absorbance is an aldamine formed by the pyridoxal 5′-phosphate-Lys276 Schiff base with the distal nitrogen of His465, the penultimate residue in the GadB sequence. Herein we show that His465 has a massive influence on the equilibrium between active and inactive forms, the former being favored when this residue is absent. His465 contributes with n ≈ 2.5 to the overall cooperativity of the system. The residual cooperativity (n ≈ 3) is associated with the conformational changes still occurring at the N-terminal ends regardless of the mutation. His465, dispensable for the cooperativity that affects enzyme activity, is essential to include the conformational change of the N termini into the cooperativity of the whole system. In the absence of His465, a 330-nm absorbing species appears, with fluorescence emission spectra more complex than model compounds and consisting of two maxima at 390 and 510 nm. Because His465 mutants are active at pH well above 5.7, they appear to be suitable for biotechnological applications.

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis.

Significance Autoimmune type 1 diabetes is characterized by the formation of self-reactive antibodies. A prevalent human autoantigen is glutamate decarboxylase (GAD)65, a highly predictive marker that can precede the emergence of disease by up to several years. Intriguingly, the closely related isoform GAD67 is not immunogenic. What are the determinants of the unique self-reactivity of GAD65 vs. GAD67? We show that, unlike GAD67, GAD65 is highly flexible and exists in multiple structural forms. We show that self-antibodies bind differentially to these GAD65 forms. These properties may be an undesirable consequence of conformational flexibility necessary for enzyme function. Our findings, thus, provide insights into how structural flexibility governs protein immunogenicity in autoimmune diabetes and have implications for therapeutic antibody and vaccine design. The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5′-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5′-phosphate–binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase

Abstract The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.

Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

Herein, biochemical and spectroscopic properties of GadB fromBrucella microti (BmGadB), aBrucella species which possesses GDAR, are described.B. microti belongs to a group of lately described and atypical brucellae that possess functionalgadB andgadC genes, unlike the most well‐known “classical”Brucella species, which include important human pathogens.BmGadB is hexameric at acidic pH. The pH‐dependent spectroscopic properties and activity profile, combined within silico sequence comparison withE. coli GadB (EcGadB), suggest thatBmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests thatBmGadB does not undergo membrane recruitment at acidic pH, which was observed inEcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved.

The yhiM gene codes for an inner membrane protein involved in GABA export in Escherichia coli

In order to survive the exposure to acid pH, Escherichia coli activates molecular circuits leading from acid tolerance to extreme acid resistance (AR). The activation of the different circuits involves several global and specific regulators affecting the expression of membrane, periplasmic and cytosolic proteins acting at different levels to dampen the harmful consequences of the uncontrolled entry of protons intracellularly. Many genes coding for the structural components of the AR circuits (protecting from pH ≤ 2.5) and their specific transcriptional regulators cluster in a genomic region named AFI (acid fitness island) and respond in the same way to global regulators (such as RpoS and H-NS) as well as to anaerobiosis, alkaline, cold and respiratory stresses, in addition to the acid stress. Notably some genes coding for structural components of AR, though similarly regulated, are non-AFI localised. Amongst these the gadBC operon, coding for the major structural components of the glutamate-based AR system, and the ybaS gene, coding for a glutaminase required for the glutamine-based AR system. The yhiM gene, a non-AFI gene, appears to belong to this group. We mapped the transcription start of the 1.1 kb monocistronic yhiM transcript: it is an adenine residue located 22 nt upstream a GTG start codon. By real-time PCR we show that GadE and GadX equally affect the expression of yhiM under oxidative growth conditions. While YhiM is partially involved in the RpoS-dependent AR, we failed to detect a significant involvement in the glutamate- or glutamine-dependent AR at pH ≤ 2.5. However, when grown in EG at pH 5.0, the yhiM mutant displays impaired GABA export, whereas when YhiM is overexpressed, an increases of GABA export in EG medium in the pH range 2.5–5.5 is observed. Our data suggest that YhiM is a GABA transporter with a physiological role more relevant at mildly acidic pH, but not a key component of AR at pH < 2.5.

The Glutaminase-Dependent System Confers Extreme Acid Resistance to New Species and Atypical Strains of Brucella

Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad)-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate (Glu)/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the “classical” species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q), relying on the deamination of glutamine (Gln) into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA)-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH ≤2.5), in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC–glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis.