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

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

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

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