Discovery of serum biomarkers for diagnosis of tuberculosis by NMR metabolomics including cross-validation with a second cohort.

BACKGROUND: Tuberculosis (TB) is a disease with worldwide presence and a major cause of death in several developing countries. Current diagnostic methodologies often lack specificity and sensitivity, whereas a long time is needed to obtain a conclusive result. METHODS: In an effort to develop better diagnostic methods, this study aimed at the discovery of a biomarker signature for TB diagnosis using a Nuclear Magnetic Resonance based metabolomics approach. In this study, we acquired 1H NMR spectra of blood serum samples of groups of healthy subjects, individuals with latent TB and of patients with pulmonary and extra-pulmonary TB. The resulting data were treated with uni- and multivariate statistical analysis. RESULTS: Six metabolites (inosine, hypoxanthine, mannose, asparagine, aspartate and glutamate) were validated by an independent cohort, all of them related with metabolic processes described as associated with TB infection. CONCLUSION: The findings of the study are according with the WHO Target Product Profile recommendations for a triage test to rule-out active TB.

Active and prospective latent tuberculosis are associated with different metabolomic profiles: clinical potential for the identification of rapid and non-invasive biomarkers.

Although 23% of world population is infected with Mycobacterium tuberculosis (M. tb), only 5-10% manifest the disease. Individuals surely exposed to M. tb that remain asymptomatic are considered potential latent TB (LTB) cases. Such asymptomatic M. tb.-exposed individuals represent a reservoir for active TB cases. Although accurate discrimination and early treatment of patients with active TB and asymptomatic M. tb.-exposed individuals are necessary to control TB, identifying those individuals at risk of developing active TB still remains a tremendous clinical challenge. This study aimed to characterize the differences in the serum metabolic profile specifically associated to active TB infected individuals or to asymptomatic M. tb.-exposed population. Interestingly, significant changes in a specific set of metabolites were shared when comparing either asymptomatic house-hold contacts of active TB patients (HHC-TB) or active TB patients (A-TB) to clinically healthy controls (HC). Furthermore, this analysis revealed statistically significant lower serum levels of aminoacids such as alanine, lysine, glutamate and glutamine, and citrate and choline in patients with A-TB, when compared to HHC-TB. The predictive ability of these metabolic changes was also evaluated. Although further validation in independent cohorts and comparison with other pulmonary infectious diseases will be necessary to assess the clinical potential, this analysis enabled the discrimination between HHC-TB and A-TB patients with an AUC value of 0.904 (confidence interval 0.81-1.00, p-value < 0.0001). Overall, the strategy described in this work could provide a sensitive, specific, and minimally invasive method that could eventually be translated into a clinical tool for TB control.

Isolation and characterization of an exopolysaccharide-producing Leuconostoc citreum strain from artisanal cheese.

High molar mass exopolysaccharides (EPS) produced from sucrose by lactic acid bacteria (LAB) are of great interest as natural additives to use in foods, medical and pharmaceutical industry. This study aimed to identify the EPS produced by Leuconostoc citreum L3C1E7 isolated from Pico cheese and characterize the strain for technological and probiotic potential. Purified EPS was isolated from the culture of L. citreum L3C1E7 by ethanol precipitation, with a yield of 520 mg ml-1 . The EPS-producing strain had a mucoid phenotype and average molecular weight of 5·88 × 106  Da. The structural characterization of the purified EPS was determined by 1 H, 13 C and two-dimensional NMR spectroscopy. EPS was composed of alternating α-(1→6)-linked and α-(1→3)-linked D-glucopyranyl units, suggesting the existence of an alternan. The strain was slow acidifying, produced diacetyl and displayed high esterase/lipase and aminopeptidase activities, which promote the desirable flavours in dairy products. Moreover, L. citreum showed moderate resistance to the adverse conditions of the gastrointestinal (GI) tract and high adhesion to GI cells. This work provides a better understanding of EPS produced by L. citreum and the potential application of EPS-producing strain in food and/or as a probiotic culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Some LAB strains are known to use extracellular glycoside-hydrolase enzymes for synthesizing a diversity of exopolysaccharides (EPS) with potential application as natural additives to foods. Previous studies have identified an EPS-producing Leuconostoc citreum strain with immunomodulatory properties. This work provides a better understanding of EPS produced by this strain and the potential application of the strain in food fermentation and/or as a probiotic culture.

NMR structure of Desulfovibrio gigas rubredoxin: a model for studying protein stabilization by compatible solutes.

Rubredoxins are small, soluble proteins that display a wide variation in thermostability, despite having a high degree of sequence similarity They also vary in the extent to which they are stabilized by solutes such as diglycerol phosphate. Hence, they provide excellent models for studying the mechanisms of thermostabilization. Nuclear magnetic resonance (NMR) spectroscopy can be used to investigate interactions between molecules, as well as subtle changes in conformation in solution, and also provides a means to measure protein stability. The assignment of the proton NMR spectrum of the zinc rubredoxin from Desulfovibrio gigas is presented, together with its structure in solution. The stabilizing effect of diglycerol phosphate on rubredoxin is demonstrated and assessed by determining selected amide proton exchange rates; diglycerol phosphate at 100 mM concentration caused an additional structural stabilization of 1.2 +/-0.4 kJ/mol. The pattern of effects on the exchange rates is discussed in relation to the protein structure.

Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus.

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

Effects of temperature, salinity, and medium composition on compatible solute accumulation by thermococcus spp

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identified as beta-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered compound, T. litoralis accumulated mannosylglycerate, aspartate, alpha-glutamate, di-myo-inositol-1,1'(3,3')-phosphate, hydroxyproline, and trehalose. The hydroxyproline and beta-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1'(3,3')-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated alpha-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of alpha-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1'(3,3')-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo-inositol-1,1'(3,3')-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.