Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants

Fructan Metabolism in A. tequilana Weber Blue Variety along Its Developmental Cycle in the Field

Fructan, as reserve carbohydrate, supplies energy needs during vegetative development, thereby exhibiting variations in its content and composition. Fructan metabolism in Agave tequilana Blue variety from 2- to 7-year-old plants was analyzed in this work. Soluble carbohydrates were determined at all ages. Fructan (328–711 mg/g), sucrose (14–39 mg/g), fructose (11–20 mg/g), glucose (4–14 mg/g), and starch (0.58–4.98 mg/g) were the most abundant carbohydrates. Thin-layer chromatography exhibited that 2–5-year-old plants mainly stored fructooligosaccharides, while 6–7-year-old plants mainly contained long-chain fructans. The fructan degree of polymerization (DP) increased from 6 to 23 throughout plant development. The 7-year-old plants mainly stored highly branched agavins. Partially methylated alditol acetate analyzed by gas chromatography–mass spectrometry reveals that fructan molecular structures became more complex with plant age. For the first time, we report the presence of a large number of DP3 (seven forms), DP4 (eight forms), and DP5 (six forms) isomers for agave fructans. Overall, fructan metabolism in A. tequilana displays changes in its soluble carbohydrates, DP, type, and fructan structures stored, along its developmental cycle in the field

Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential of that reported for C. albicans. In addition, we propose that purified cell wall mannans can be used as antagonist to block specific receptors on innate immune cells

Schematic representation of the genomic structures of <i>A. tequilana</i> Fructosyltransferases and Invertases.

<p>Introns are represented by lines and exons with solid boxes. Exons are distinguished by Roman numerals from left to right. Only <i>AtqCwinv-1</i> shows a distinct pattern of exon/intron number and organization. Drawings are to scale and scale bar represents 250 bp.</p

qRT-PCR expression profiles of <i>A. tequilana</i> fructosyltransferase and invertase genes in different plant tissues and developmental stages.

<p>A. <i>Atq1-SST-2,</i> B. <i>AtqVinv-1</i>, C. <i>Atq6G-FFT-1</i>, D. <i>AtqCwinv-1.</i> S-stem, BL-Base of leaf and ML-Mid-leaf of 1 and 3, year old plants (vegetative stage) and 5 and 7 year old plants (Post-reproductive stage). Photographic examples of tissues sampled are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035878#pone.0035878.s001" target="_blank">Figure S1</a>.</p

Unrooted tree of selected fructosyltransferases, fructan exohydrolases and invertases from monocotyledonous and dicotyledonous plants.

<p>A: branch containing cell wall invertases and FEHs, B: branch containing vacuolar invertases and fructosyltransferases, b1/b2-monocotyledons, b3-dicotyledons. <i>A. tequilana</i> fructosyltransferases and invertases are indicated with a diamond. Accession numbers are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035878#pone.0035878.s005" target="_blank">Table S1</a>.</p

Alignment of deduced amino acid sequences of fructosyltransferases and Invertases of <i>A. tequilana</i>.

<p>Asterisks, colons and periods indicate identical residues, conserved substitutions, and semi-conserved substitutions, respectively. Putative initiation points of the large and small subunits are arrowed. Potential glycosylation sites are underlined. The β-fructosidase motif, RDP motif and the cysteine catalytic site are boxed. A sucrose-donor substrate motif is shown as a stippled box. Predicted leader sequence cleavage points are shaded in grey. Differences between <i>Atq1-SST-1</i> and SSTAg are shown in italics.</p

Activity of recombinant 6G-FFT-1 protein.

<p>HPAEC chromatograms of the products obtained by the reactions supplied with: A. 100 mM sucrose. B. 50 mM 1-kestose C. both 100 mM sucrose and 50 mM 1-kestose D-amplification of the chromatogram in C. Abbreviations G-glucose, F-fructose, S-sucrose, 1-K-1-kestose, Neo-Neokestose, Nys-nystose, 4c-1^F,6^G-Di-β-D-fructofuranosylsucrose. Unidentified products are indicated by asterisks.</p

Activities of recombinant AtqCwinv-1 and Atq1-SST-2 proteins.

<p>A. Time-course of reaction products when 100 mM sucrose is supplied as substrate for AtqCinv-1. B. HPAEC chromatograms of the products when 100 mM 1-kestose was supplied as substrate for AtqCinv-1. C. Time course of reaction products of Atq1-SST2 when 100 mM sucrose is supplied as substrate and D. HPAEC chromatograms of the products of the Atq1-SST2 reactions when 100 mM sucrose was supplied as substrate. G-glucose, F-fructose, S-sucrose, 1-K-1-kestose, Neo-Neokestose, Nys-nystose.</p

Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

Sporothrix schenckii sensu stricto and S. brasiliensis are usually associated to sporotrichosis, a subcutaneous mycosis worldwide distributed. Comparative analyses between these two species indicate they contain genetic and physiological differences that are likely to impact the interaction with host cells. Here, we study the composition of the cell wall from conidia, yeast-like cells and germlings of both species and found they contained the same sugar composition. The carbohydrate proportion in the S. schenckii sensu stricto wall was similar across the three cell morphologies, with exception in the chitin content, which was significantly different in the three morphologies. The cell wall from germlings showed lower rhamnose content and higher glucose levels than other cell morphologies. In S. brasiliensis, the wall sugars were constant in the three morphologies, but glucose was lower in yeast-like cells. In S. schenckii sensu stricto cells most of chitin and β1,3-glucan were underneath wall components, but in S. brasiliensis germlings, chitin was exposed at the cell surface, and β1,3-glucan was found in the outer part of the conidia wall. We also compared the ability of these cells to stimulate cytokine production by human peripheral blood mononuclear cells. The three S. schenckii sensu stricto morphologies stimulated increased levels of pro-inflammatory cytokines, when compared to S. brasiliensis cells; while the latter, with exception of conidia, stimulated higher IL-10 levels. Dectin-1 was a key receptor for cytokine production during stimulation with the three morphologies of S. schenckii sensu stricto, but dispensable for cytokine production stimulated by S. brasiliensis germlings. TLR2 and TLR4 were also involved in the sensing of Sporothrix cells, with a major role for the former during cytokine stimulation. Mannose receptor had a minor contribution during cytokine stimulation by S. schenckii sensu stricto yeast-like cells and germlings, but S. schenckii sensu stricto conidia and S. brasiliensis yeast-like cells stimulated pro-inflammatory cytokines via this receptor. In conclusion, S. brasiliensis and S. schenckii sensu stricto, have similar wall composition, which undergoes changes depending on the cell morphology. These differences in the cell wall composition, are likely to influence the contribution of immune receptors during cytokine stimulation by human monocytes