Allostery and protein plasticity: the keystones for bacterial signaling and regulation.

Bacteria sense intracellular and environmental signals using an array of proteins as antennas. The information is transmitted from such sensory modules to other protein domains that act as output effectors. Sensor and effector can be part of the same polypeptide or instead be separate diffusible proteins that interact specifically. The output effector modules regulate physiologic responses, allowing the cells to adapt to the varying conditions. These biological machineries are known as signal transduction systems (STSs). Despite the captivating architectural diversity exhibited by STS proteins, a universal feature is their allosteric regulation: signal binding at one site modifies the activity at a physically distant site. Allostery requires protein plasticity, precisely encoded within their 3D structures, and implicating programmed molecular motions. This review summarizes how STS proteins connect stimuli to specific responses by exploiting allostery and protein plasticity. Illustrative examples spanning a wide variety of protein folds will focus on one- and two-component systems (TCSs). The former encompass the entire transmission route within a single polypeptide, whereas TCSs have evolved as separate diffusible proteins that interact specifically, sometimes including additional intermediary proteins in the pathway. Irrespective of their structural diversity, STS proteins are able to modulate their own molecular motions, which can be relatively slow, rigid-body movements, all the way to fast fluctuations in the form of macromolecular flexibility, thus spanning a continuous protein dynamics spectrum. In sum, STSs rely on allostery to steer information transmission, going from simple two-state switching to rich multi-state conformational order/disorder transitions.

The crystal structure of the malic enzyme from Candidatus Phytoplasma reveals the minimal structural determinants for a malic enzyme.

Phytoplasmas are wall-less phytopathogenic bacteria that produce devastating effects in a wide variety of plants. Reductive evolution has shaped their genome, with the loss of many genes, limiting their metabolic capacities. Owing to the high concentration of C4 compounds in plants, and the presence of malic enzyme (ME) in all phytoplasma genomes so far sequenced, the oxidative decarboxylation of L-malate might represent an adaptation to generate energy. Aster yellows witches'-broom (Candidatus Phytoplasma) ME (AYWB-ME) is one of the smallest of all characterized MEs, yet retains full enzymatic activity. Here, the crystal structure of AYWB-ME is reported, revealing a unique fold that differs from those of `canonical' MEs. AYWB-ME is organized as a dimeric species formed by intertwining of the N-terminal domains of the protomers. As a consequence of such structural differences, key catalytic residues such as Tyr36 are positioned in the active site of each protomer but are provided by the other protomer of the dimer. A Tyr36Ala mutation abolishes the catalytic activity, indicating the key importance of this residue in the catalytic process but not in the dimeric assembly. Phylogenetic analyses suggest that larger MEs (large-subunit or chimeric MEs) might have evolved from this type of smaller scaffold by gaining small sequence cassettes or an entire functional domain. The Candidatus Phytoplasma AYWB-ME structure showcases a novel minimal structure design comprising a fully functional active site, making this enzyme an attractive starting point for rational genetic design.

What Makes a Bacterial Species Pathogenic?: Comparative Genomic Analysis of the Genus Leptospira

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts

STRUCTURAL VIROLOGY. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography.

Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus. CA is organized as hexamers that deviate substantially from sixfold symmetry, yet adjust to make two-dimensional pseudohexagonal arrays that mimic mature retroviral cores. Intra- and interhexameric quasi-equivalent contacts are uncovered, with flexible trimeric lateral contacts among hexamers, yet preserving very similar dimeric interfaces making the lattice. The conformation of each capsid subunit in the hexamer is therefore dictated by long-range interactions, revealing how the hexamers can also assemble into closed core particles, a relevant feature of retrovirus biology.

Volume of interest-based [18F]fluorodeoxyglucose PET discriminates MCI converting to Alzheimer's disease from healthy controls. A European Alzheimer's Disease Consortium (EADC) study.

An emerging issue in neuroimaging is to assess the diagnostic reliability of PET and its application in clinical practice. We aimed at assessing the accuracy of brain FDG-PET in discriminating patients with MCI due to Alzheimer's disease and healthy controls. Sixty-two patients with amnestic MCI and 109 healthy subjects recruited in five centers of the European AD Consortium were enrolled. Group analysis was performed by SPM8 to confirm metabolic differences. Discriminant analyses were then carried out using the mean FDG uptake values normalized to the cerebellum computed in 45 anatomical volumes of interest (VOIs) in each hemisphere (90 VOIs) as defined in the Automated Anatomical Labeling (AAL) Atlas and on 12 meta-VOIs, bilaterally, obtained merging VOIs with similar anatomo-functional characteristics. Further, asymmetry indexes were calculated for both datasets. Accuracy of discrimination by a Support Vector Machine and the AAL VOIs was tested against a validated method (PALZ). At the voxel level SMP8 showed a relative hypometabolism in the bilateral precuneus, and posterior cingulate, temporo-parietal and frontal cortices. Discriminant analysis classified subjects with an accuracy ranging between .91 and .83 as a function of data organization. The best values were obtained from a subset of 6 meta-VOIs plus 6 asymmetry values reaching an area under the ROC curve of .947, significantly larger than the one obtained by the PALZ score. High accuracy in discriminating MCI converters from healthy controls was reached by a non-linear classifier based on SVM applied on predefined anatomo-functional regions and inter-hemispheric asymmetries. Data pre-processing was automated and simplified by an in-house created Matlab-based script encouraging its routine clinical use. Further validation toward nonconverter MCI patients with adequately long follow-up is needed.

Functional imaging in pre-motor Parkinson's disease.

Several non motor symptoms (NMS) can precede the onset of the classical motor Parkinson's disease (PD) syndrome. The existence of pre-motor and even pre-clinical PD stages has been proposed but the best target population to be screened to disclose PD patients in a pre-clinical, thus asymptomatic, stage is still matter of debate. The REM sleep behavior disorder (RBD) often affects PD patients at different stages of the disease and could precede the onset of motor symptoms by several years. However, RBD could also precede other synucleinopathies (namely, dementia with Lewy bodies and multisystem atrophy), and less frequently could be related to other neurological conditions or remain idiopathic. Moreover, not all PD patients exhibit RBD. Despite these caveats, RBD probably represents the best feature to disclose pre-motor PD patients given its high-risk of developing a full motor syndrome. Other clinical clues in the premotor stages of PD undergoing active investigation include hyposmia, depression, and autonomic dysfunction. Effective biomarkers are needed in order to improve the diagnostic accuracy in the pre-motor stage of PD, to monitor disease progression and to plan both pharmacological and non-pharmacological intervention. Functional imaging, in particular radionuclide methodologies, has been often used to investigate dopaminergic and non-dopaminergic features as well as cortical functioning in patients with RBD in its idiopathic form (iRBD) and/or associated with PD. Recently, new tracers to image α-synuclein pathologies are under development. Functional imaging in pre-motor PD, and in particular in iRBD, could improve our knowledge about the underlying mechanisms and the neurodegenerative progress of PD.

Trafficking and homing of systemically administered stem cells: the need for appropriate analysis tools of radionuclide images.

AIM: Despite its enormous relevance, homing of hematopoietic stem cells (SCs) remains relatively uncertain due to the limitations of measuring small number of systemically administered cells in the different organs. Despite its high sensitivity, radionuclide detection has been relatively underutilized to this purpose since it cannot differentiate hematopietic SCs recruited by target tissues from those circulating in the blood pool. Our study aims to verify the potential of tracer kinetic approaches in estimating the recruitment of labeled SCs after their systemic administration. METHODS: Twenty-four Lewis rats underwent administration of 2 millions cells labeled with 37 MBq of 99mTc-exametazime. Animals were divided into 2 groups according to administered cells: hematopoietic SCs or cells obtained from a line of rat hepatoma. Cell injection was performed during a planar dynamic acquisition. Regions of interest were positioned to plot time activity curves on heart, lungs, liver and spleen. Blood cell clearance was evaluated according to common stochastic analysis approach. Either fraction of dose in each organ at the end of the experiment or computing the slope of regression line provided by Patlak or Logan graphical approach estimated cell recruitment. At the end of the study, animals were sacrificed and the number of cells retained in the same organs was estimated by in vitro counting. RESULTS: Cell number, documented by the dose fraction retained in each organ at imaging was consistently higher with respect to the "gold standard" in vitro counting in all experiments. An inverse correlation was observed between degree of overestimation and blood clearance of labeled cells (r=-0.56, P<0.05). Logan plot analysis consistently provided identifiable lines, whose slope values closely agreed with the "in vitro" estimation of hepatic and splenic cell recruitment. CONCLUSION: The simple evaluation of organ radioactivity concentration does not provide reliable estimates of local recruitment of systemically administered cells. Yet, the combined analysis of temporal trends of tracer (cell) tissue accumulation and blood clearance can provide quantitative estimations of cell homing in the different organs.

Liver transplantation in systemic lupus erythematosus: case report and review of the literature.

Severe liver involvement requiring liver transplantation is a rare complication in systemic lupus erythematosus (SLE), but very few cases have been reported. We describe a 39-year-old woman with SLE who underwent successful liver transplantation due to acute liver failure. The patient persisted without reactivation of SLE and with good long-term survival. Management and diagnosis considerations are reviewed.

Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity.

Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.

The tyrosine aminotransferase from Trypanosoma rangeli: sequence and genomic characterization.

The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates.

Structural basis of sialyltransferase activity in trypanosomal sialidases.

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase, which is involved in key aspects of parasite-host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic beta-propeller fold tightly associated with a lectin-like domain. Comparison with the modeled structure of T.cruzi trans-sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure-based drug design.

Trypanosoma rangeli sialidase: cloning, expression and similarity to T. cruzi trans-sialidase.

Sialidases are hydrolytic enzymes present from virus to higher eukaryotes, catalyzing the removal of sialic acid from glycoconjugates. Some protozoa Trypanosomatidae secrete high levels of sialidase into the medium. We have now purified the secreted sialidase from Trypanosoma rangeli. Its N-terminal sequence reveals 100% identity with the corresponding region of the trans-sialidase from T. cruzi. Trans-sialidase, although homologous to viral and bacterial sialidases, displays a novel sialyltransferase activity and is involved in host cell invasion. Several homologous transsialidase-like genes were cloned from genomic DNA of T. rangeli, and grouped in three subfamilies. Active sialidase-encoding genes were found in one of them. The recombinant sialidase shows similar properties to those of the native enzyme, including undetectable trans-sialidase activity. Nevertheless, it has an overall identity of 68.9% with the catalytic domain of T. cruzi trans-sialidase, increasing to 86.7% admitting conservative substitutions. Only three other eukaryotic sialidases have been previously cloned, none of them showing significant homology to trans-sialidase. The isolation of a highly similar sialidase is relevant to further identify the molecular determinants allowing trans-sialidase activity. As a first approach, chimeric constructs between sialidase and trans-sialidase were generated, one of them rendering a sialidase with three times lower Km than the natural enzyme.

Medium scale production and purification to homogeneity of a recombinant trans-sialidase from Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas' disease, presents an enzyme that catalyzes the transfer of sialic acid among glycoproteins and glycolipids known as trans-sialidase (TS), displaying some interesting features: 1) It differs from all other eucaryotic sialyltransferases, both kinetically and in substrate specificity and 2) it is involved in the parasite's mechanism of mammalian host cell invasion. We report here the production and purification to homogeneity of an enzymatically active recombinant TS (rTS) lacking the C-terminal amino acid repeats, using iminodiacetic metal affinity chromatography. Initial ratios of non-fusion recombinant versus total protein were very low in several expression systems tested, mainly due to high degradation rate. However, after purifying 1,330 times, we were able to obtain an essentially homogeneous preparation of rTS with a final yield of 29%. After minor changes, a modified protocol for a medium scale production was designed obtaining 0.5 mg of homogeneous rTS per liter of bacterial culture. The purified rTS behaved as a homogeneous protein in silver-stained denaturing gels, isoelectrofocusing and N-terminal sequencing having identical pH and temperature optima as the natural enzyme. Conditions to keep the rTS for long periods without a significant loss of activity were identified.

Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi.

The gene(s) encoding the Trypanosoma cruzi shed-acute-phase-antigen (SAPA) has a 5' end encoding a region containing two totally and two partially conserved Ser-X-Asp-X-Gly-X-Thr-Trp motifs which are present in bacterial neuraminidases, and a 3' end encoding tandemly repeated units of 12 amino acids. It is now reported that 54-87% of the total neuraminidase activity present in the parasite could be immunoprecipitated with polyclonal or monoclonal antibodies against the repeated amino acid units of SAPA. These immunoprecipitates also had greater than 80% of the trans-sialidase activity of the parasite. SAPA used sialyllactose, fetuin and 4-methylumbelliferyl-sialic acid as substrate donors. In the presence of a suitable acceptor molecule (lactose) the sialic acid residues were transferred to the disaccharide, whereas in the absence of acceptors the residues were transferred to water. If relatively inefficient acceptors (maltose or cellobiose) were added to the incubation mixtures, the sialic acid units were transferred both to the disaccharides and to water. It is concluded that a major T. cruzi antigen has both the trans-sialidase and the neuraminidase activities of the parasite. Both activities are probably located on the N-terminus of SAPA since antibodies directed against the C-terminus, which contains the repeated amino acid units, do not affect the enzymatic activities.