Balouz V, Lopez R, Conte N, Abal M, Ducrey I, et al.
PLoS neglected tropical diseases •
Chagas disease (ChD), caused by the parasitic protozoan Trypanosoma cruzi, is a lifelong, neglected tropical disease with substantial medical and socioeconomic impact. Despite this situation, currently available diagnostic and therapeutic methods display serious limitations. A promising strategy to improve ChD serodiagnosis involves targeting parasite carbohydrate antigens, particularly the α-galactosyl-rich mucins that coat the surface of bloodstream trypomastigotes (tGPI-mucins). Here, we present a concise and efficient protocol for the chemical synthesis of a tGPI-mucin-derived glycotope, the disaccharide α-D-Galp-(1 → 3)-β-D-Galp, and its functional conjugation to different scaffolds using the squarate method. A neoglycoprotein made upon a bovine serum albumin (BSA) carrier decorated with ∼38 units of the disaccharide, termed BSA-Di, was interrogated with sera of chronic ChD patients and healthy individuals from Argentina using an in-house enzyme-linked immunosorbent assay (ELISA). BSA-Di exhibited excellent sensitivity and effectively discriminated between ChD-positive and negative sera with high accuracy (AUC = 0.905), though its specificity was partially affected by cross-reactivity of some non-ChD sera containing natural α-Gal antibodies. Conjugation of α-D-Galp-(1 → 3)-β-D-Galp to T. cruzi antigenic peptides, instead of BSA, corroborated these findings and enabled the generation of bivalent ChD diagnostic reagents combining glycan- and peptide-based epitopes. Overall, our results identify α-D-Galp-(1 → 3)-β-D-Galp as a robust and reliable biomarker of T. cruzi infection. The methodologies and tools described here, together with optimized derivatives, are expected to positively impact ChD serological applications.
Abal M, Balouz V, Lopez R, Giorgi ME, Marino C, et al.
PLoS neglected tropical diseases •
Proper evaluation of therapeutic responses in Chagas disease is hampered by the prolonged persistence of antibodies to Trypanosoma cruzi measured by conventional serological tests and by the lack of sensitivity of parasitological tests. Previous studies indicated that tGPI-mucins, an α-Gal (α-d-Galp(1→3)-β-d-Galp(1→4)-d-GlcNAc)-rich fraction obtained from T. cruzi trypomastigotes surface coat, elicit a strong and protective antibody response in infected individuals, which disappears soon after successful treatment. The cost and technical difficulties associated with tGPI-mucins preparation, however, preclude its routine implementation in clinical settings. We herein developed a neoglycoprotein consisting of a BSA scaffold decorated with several units of a synthetic α-Gal antigenic surrogate (α-d-Galp(1→3)-β-d-Galp(1→4)-β-d-Glcp). Serological responses to this reagent, termed NGP-Tri, were monitored by means of an in-house enzyme-linked immunosorbent assay (α-Gal-ELISA) in a cohort of 82 T. cruzi-infected and Benznidazole- or Nifurtimox-treated children (3 days to 16 years-old). This cohort was split into 3 groups based on the age of patients at the time of treatment initiation: Group 1 comprised 24 babies (3 days to 5 months-old; median = 26 days-old), Group 2 comprised 31 children (7 months to 3 years-old; median = 1.0-year-old) and Group 3 comprised 26 patients (3 to 16 years-old; median = 8.4 years-old). A second, control cohort (Group 4) included 39 non-infected infants (3 days to 5 months-old; median = 31 days-old) born to T. cruzi-infected mothers. Despite its suboptimal seroprevalence (58.4%), α-Gal-ELISA yielded shorter median time values of negativization (23 months [IC 95% 7 to 36 months] vs 60 months [IC 95% 15 to 83 months]; p = 0.0016) and higher rate of patient negative seroconversion (89.2% vs 43.2%, p < 0.005) as compared to conventional serological methods. The same effect was verified for every Group, when analyzed separately. Most remarkably, 14 out of 24 (58.3%) patients from Group 3 achieved negative seroconversion for α-Gal-ELISA while none of them were able to negativize for conventional serology. Detailed analysis of patients showing unconventional serological responses suggested that, in addition to providing a novel tool to shorten follow-up periods after chemotherapy, the α-Gal-ELISA may assist in other diagnostic needs in pediatric Chagas disease. The tools evaluated here provide the cornerstone for the development of an efficacious, reliable, and straightforward post-therapeutic marker for pediatric Chagas disease.
Montoya AL, Gil ER, Vinales I, Estevao IL, Taboada P, et al.
Carbohydrate research •
The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfβ conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfβ1,3Manpα(CH)SH (G31) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.
Zanuzzi CN, Gomez Castro G, Hernández R, Tozzi R, Falcón JE, et al.
Reproduction in domestic animals = Zuchthygiene •
This article describes the carbohydrate composition of early and mature placentas from bitches, detected by lectin histochemistry. Formalin-fixed placental samples from 11 mixed-breed bitches have been assigned to the 'early' or the 'mature' placenta group, processed by the routine histological technique and labelled with a panel of 14 biotinylated lectins. The glycan distribution was almost completely preserved over pregnancy. Cytotrophoblast cells expressed high-mannose N-linked residues and N-acetyl-D-glucosamine, whereas mannose N-linked residues, N-acetyl-D-glucosamine and β- and α-D-galactose/N-acetyl-D-galactosamine have been found in the syncytiotrophoblast. The maternal and foetal endothelial binding pattern was enriched in β-D-galactose, α-D-mannose in non-bisected bi/tri-antennary N-linked complex and Galα1,3Gal-Galα1,4Gal. Both deep and superficial glands showed a great variety of glycoconjugates, such as D-mannose, D-glucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, galactose, N-acetylneuraminic acid and fucose. Most results in this study were consistent with those previously reported in canine and feline mature placentas; here, early placentas have been analysed and the lectin binding pattern of mature placentas has been further described. These studies on canine placentas contribute to detecting and understanding glycome changes in pathophysiological conditions.
Montoya AL, Gil ER, Heydemann EL, Estevao IL, Luna BE, et al.
Molecules (Basel, Switzerland) •
Chagas disease (CD) can be accurately diagnosed by detecting in patients' blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method's limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal β-galactofuranosyl (β-Gal) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients' sera react specifically with synthetic β-Gal-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of GIPL-derived Galβ1,3Manα-(CH)SH (glycan ) and Galβ1,3Manα1,2-[Galβ1,3]Manα-(CH)SH (glycan ); and (b) preparation of neoglycoproteins and , and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galβ1,3Manα1,2-[Galβ1,3]Manα is an immunodominant glycotope and that could potentially be used as a novel CCD biomarker.
Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal β-d-Galp unit in the host glycoconjugate to terminal β-d-Galp units in the parasite mucins to construct the d-NeuNAc(α2→3)β-d-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-d-Galp is only present in the infective stage whereas β-d-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-d-Galp nor d-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal β-d-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-d-Galp(1→3)-[β-d-Galp(1→6)]-d-Galp, the smallest structure containing both, the antigenic d-Galp(α1→3)-d-Galp unit and the sialic acid-acceptor β-d-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by β-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3'-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-d-NeuNAc(2→3)-β-d-Galp(1→6)-[α-d-Galp(1→3)]-β-d-Galp, was purified and characterized.
Carrera-Silva EA, Silvestroni A, LeBlanc JG, Piard JC, Savoy de Giori G, et al.
Current microbiology •
Alpha-galactosidase (alpha-Gal) enzyme, which is encoded by the melA gene hydrolyzes alpha-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These alpha-galacto-oligosaccharides (alpha-GOS), which are found in large quantities in vegetables, such as soy, can cause gastrointestinal disorders in sensitive individuals because monogastric animals (including humans) do not posses alpha-Gal in the gut. The use of microbial alpha-Gal is a promising alternative to eliminate alpha-GOS in soy-derived products. Using degenerate primers, the melA gene from Lactobacillus (L.) fermentum CRL722 was identified. The complete genomic sequence of melA (2223 bp), and of the genes flanking melA, were obtained using a combination of polymerase chain reaction-based techniques, and showed strong similarities with the alpha-Gal gene of thermophilic microorganisms. The alpha-Gal gene from L. fermentum CRL722 was cloned and the protein purified from cell-free extracts of the native and recombinant strains using various techniques (ion exchange chromatography, salt precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ultra-filtration); Its main biochemical properties were determined. The enzyme was active at moderately high temperatures (55 degrees C) and stable at wide ranges of temperatures and pH. The thermostable alpha-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of alpha-GOS found in soy products. The complete melA gene could also be inserted in other micro-organisms, that can survive in the harsh conditions of the gut to degrade alpha-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value.
The influence of environmental pH on biological activity of Bifidobacterium longum CRL 849 grown in MRS-raffinose was evaluated. At pH 6.0, 5.5 and 5.0, raffinose was completely consumed by this microorganism, showing different consumption rates at each pH value (between 3.03 and 0.76 mmol l(-1) h(-1)). At pH 4.5, the growth was lowest. The removal of raffinose was due to the alpha-galactosidase (alpha-gal) activity of this bifidobacteria, which was highest at pH 6.0-5.5 (1,280-1,223 mU ml(-1)). The production of beta-glucosidase (beta-glu) showed a similar pattern to alpha-gal activity with major values. The yield of organic acids produced during raffinose consumption was also highest at pH 6.0-5.5. The results of this study will allow the selection of the optimum growth conditions of B. longum CRL 849, with elevated levels of alpha-gal to be used in the reduction of nondigestible alpha-oligosaccharide in soy products and beta-glu activities involved in isoflavone conversion to bioactive forms when used as starter culture.
In this study, the behaviour of Lactobacillus fermentum CRL 722 and CRL 251 were evaluated under different pH conditions (pH 6.0, 5.5, 5.0, 4.5) and without pH control. Growth was similar under all conditions assayed except at pH 4.5. These microorganisms were able to eliminate raffinose, a nondigestible alpha-oligosaccharide (NDO) found in soy products, showing a consumption rate of 0.25 g l(-1) h(-1) (pH 6.0-5.0). The removal of raffinose was due to the high alpha-galactosidase (alpha-gal) activities of these lactic acid bacteria, which was highest at pH 5.5 (5.0 U/ml). The yield of organic acids produced during raffinose consumption was also highest at this pH. The results of this study will allow selection of the optimum growth conditions of L. fermentum with elevated levels of alpha-gal to be used in the reduction of NDO in soy products when used as starter cultures.
LeBlanc JG, Garro MS, Silvestroni A, Connes C, Piard JC, et al.
Journal of applied microbiology •
Consumption of soya-derived products has been hampered by the presence of alpha-galactooligosaccharides (alpha-GOS) because mammals lack pancreatic alpha-galactosidase (alpha-Gal) which is necessary for their hydrolysis. These sugars thus reach the large intestine causing gastrointestinal disorders in sensitive individuals. The use of lactic acid bacteria (LAB) expressing alpha-Gal is a promising solution for the degradation of alpha-GOS in soyamilk. The capacity of the LAB Lactobacillus fermentum CRL 722 to properly degrade alpha-GOS was studied in vitro using controlled fermentation conditions and in vivo using a rat model. Lactobacillus fermentum CRL 722 was able to grow on commercial soyamilk and completely eliminated stachyose and raffinose during fermentation because of its high alpha-Gal activity. Rats fed soyamilk fermented by this LAB had smaller caecums compared with rats fed unfermented soyamilk. Soyamilk fermentation by Lact. fermentum CRL 722 results in the reduction of alpha-GOS concentrations in soyamilk, thus eliminating possible undesirable physiological effects normally associated with its consumption. Fermentation with Lact. fermentum CRL 722 could prevent gastrointestinal disorders in sensitive individuals normally associated with the consumption of soya-based products. This LAB could thus be used in the elaboration of novel fermented vegetable products which better suit the digestive capacities of consumers.
Silvestroni A, Connes C, Sesma F, De Giori GS, Piard JC
Applied and environmental microbiology •
Alpha-galactosides are abundant sugars in legumes such as soy. Because of the lack of alpha-galactosidase (alpha-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence. To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous alpha-Gal is promising. In this framework, we characterized the melA gene for alpha-Gal in Lactobacillus plantarum. The melA gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers. The melA gene was cloned and expressed in Escherichia coli, yielding an active alpha-Gal. We show that melA is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose. Posttranscriptional regulation by the carbon source could also occur. Upstream of melA, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both alpha- and beta-galactosides in Streptococcus thermophilus. rafP is also expressed as a monocistronic mRNA. Downstream of melA, the lacL and lacM genes were identified that encode a heterodimeric beta-galactosidase. A putative galM gene identified in the same cluster suggests the presence of a galactose operon. These results indicate that the genes involved in galactoside catabolism are clustered in L. plantarum ATCC 8014. This first genetic characterization of melA and of its putative associated transporter, rafP, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues.
