The Role of IL-17-Producing Cells in Cutaneous Fungal Infections.

The skin is the outermost layer of the body and is exposed to many environmental stimuli, which cause various inflammatory immune responses in the skin. Among them, fungi are common microorganisms that colonize the skin and cause cutaneous fungal diseases such as candidiasis and dermatophytosis. The skin exerts inflammatory responses to eliminate these fungi through the cooperation of skin-component immune cells. IL-17 producing cells are representative immune cells that play a vital role in anti-fungal action in the skin by producing antimicrobial peptides and facilitating neutrophil infiltration. However, the actual impact of IL-17-producing cells in cutaneous fungal infections remains unclear. In this review, we focused on the role of IL-17-producing cells in a series of cutaneous fungal infections, the characteristics of skin infectious fungi, and the recognition of cell components that drive cutaneous immune cells.

TBL10 is required for O-acetylation of pectic rhamnogalacturonan-I in Arabidopsis thaliana.

O-Acetylated pectins are abundant in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O-acetylation of pectins are still largely unknown. In this study, we showed that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O-acetylation of pectins in Arabidopsis (Arabidopsis thaliana). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2, were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O-acetylation of RG-I, possibly as an acetyltransferase, and suggest that O-acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

Overexpression of bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase leads to enhanced photosynthesis and global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002.

Cyanobacteria fix atmospheric CO2 to biomass and through metabolic engineering can also act as photosynthetic factories for sustainable productions of fuels and chemicals. The Calvin Benson cycle is the primary pathway for CO2 fixation in cyanobacteria, algae and C3 plants. Previous studies have overexpressed the Calvin Benson cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (hereafter BiBPase), in both plants and algae, although their impacts on cyanobacteria have not yet been rigorously studied. Here, we show that overexpression of BiBPase and RuBisCO have distinct impacts on carbon metabolism in the cyanobacterium Synechococcus sp. PCC 7002 through physiological, biochemical, and proteomic analyses. The former enhanced growth, cell size, and photosynthetic O2 evolution, and coordinately upregulated enzymes in the Calvin Benson cycle including RuBisCO and fructose-1,6-bisphosphate aldolase. At the same time it downregulated enzymes in respiratory carbon metabolism (glycolysis and the oxidative pentose phosphate pathway) including glucose-6-phosphate dehydrogenase (G6PDH). The content of glycogen was also significantly reduced while the soluble carbohydrate content increased. These results indicate that overexpression of BiBPase leads to global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002, promoting photosynthetic carbon fixation and carbon partitioning towards non-storage carbohydrates. In contrast, whilst overexpression of RuBisCO had no measurable impact on growth and photosynthetic O2 evolution, it led to coordinated increase in the abundance of proteins involved in pyruvate metabolism and fatty acid biosynthesis. Our results underpin that singular genetic modifications in the Calvin Benson cycle can have far broader cellular impact than previously expected. These features could be exploited to more efficiently direct carbons towards desired bioproducts.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n , wherein n = 10 or 20]. The yields of the (SP)n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins.

The Non-Coding RNA Ncr0700/PmgR1 is Required for Photomixotrophic Growth and the Regulation of Glycogen Accumulation in the Cyanobacterium Synechocystis sp. PCC 6803.

Carbohydrate metabolism is a tightly regulated process in photosynthetic organisms. In the cyanobacterium Synechocystis sp. PCC 6803, the photomixotrophic growth protein A (PmgA) is involved in the regulation of glucose and storage carbohydrate (i.e. glycogen) metabolism, while its biochemical activity and possible factors acting downstream of PmgA are unknown. Here, a genome-wide microarray analysis of a ΔpmgA strain identified the expression of 36 protein-coding genes and 42 non-coding transcripts as significantly altered. From these, the non-coding RNA Ncr0700 was identified as the transcript most strongly reduced in abundance. Ncr0700 is widely conserved among cyanobacteria. In Synechocystis its expression is inversely correlated with light intensity. Similarly to a ΔpmgA mutant, a Δncr0700 deletion strain showed an approximately 2-fold increase in glycogen content under photoautotrophic conditions and wild-type-like growth. Moreover, its growth was arrested by 38 h after a shift to photomixotrophic conditions. Ectopic expression of Ncr0700 in Δncr0700 and ΔpmgA restored the glycogen content and photomixotrophic growth to wild-type levels. These results indicate that Ncr0700 is required for photomixotrophic growth and the regulation of glycogen accumulation, and acts downstream of PmgA. Hence Ncr0700 is renamed here as PmgR1 for photomixotrophic growth RNA 1.

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria.

BACKGROUND: Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls, lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons. RESULTS: Here we show an easy and highly efficient method for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism in cyanobacteria. Incubation of the cyanobacterial cells in the commercially available B-PER reagent for 10 min permeabilized the cells, as confirmed by the SYTOX Green staining. There was no significant change in the cell shape and no major loss of intracellular proteins was observed during the treatment. When used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at -20 °C without losing the enzyme activities. The permeabilization process and subsequent activity assays were successfully adapted to the 96-well plate system. CONCLUSIONS: An easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner.

A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.

A growing body of evidence suggests that protein-protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.

Biotechnological exploitation of microalgae.

Microalgae are a diverse group of single-cell photosynthetic organisms that include cyanobacteria and a wide range of eukaryotic algae. A number of microalgae contain high-value compounds such as oils, colorants, and polysaccharides, which are used by the food additive, oil, and cosmetic industries, among others. They offer the potential for rapid growth under photoautotrophic conditions, and they can grow in a wide range of habitats. More recently, the development of genetic tools means that a number of species can be transformed and hence used as cell factories for the production of high-value chemicals or recombinant proteins. In this article, we review exploitation use of microalgae with a special emphasis on genetic engineering approaches to develop cell factories, and the use of synthetic ecology approaches to maximize productivity. We discuss the success stories in these areas, the hurdles that need to be overcome, and the potential for expanding the industry in general.

The endo-arabinanase BcAra1 is a novel host-specific virulence factor of the necrotic fungal phytopathogen Botrytis cinerea.

The plant cell wall is one of the first physical interfaces encountered by plant pathogens and consists of polysaccharides, of which arabinan is an important constituent. During infection, the necrotrophic plant pathogen Botrytis cinerea secretes a cocktail of plant cell-wall-degrading enzymes, including endo-arabinanase activity, which carries out the breakdown of arabinan. The roles of arabinan and endo-arabinanases during microbial infection were thus far elusive. In this study, the gene Bcara1 encoding for a novel α-1,5-L-endo-arabinanase was identified and the heterologously expressed BcAra1 protein was shown to hydrolyze linear arabinan with high efficiency whereas little or no activity was observed against the other oligo- and polysaccharides tested. The Bcara1 knockout mutants displayed reduced arabinanase activity in vitro and severe retardation in secondary lesion formation during infection of Arabidopsis leaves. These results indicate that BcAra1 is a novel endo-arabinanase and plays an important role during the infection of Arabidopsis. Interestingly, the level of Bcara1 transcript was considerably lower during the infection of Nicotiana benthamiana compared with Arabidopsis and, consequently, the ΔBcara1 mutants showed the wild-type level of virulence on N. benthamiana leaves. These results support the conclusion that the expression of Bcara1 is host dependent and is a key determinant of the disease outcome.

Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex.

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

A Case Report of Mycosis Fungoides Presenting With Blister Formation.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma with a usually indolent course. Early detection is crucial for effective intervention. We present a case of a 40-year-old male with MF exhibiting blistering as a rare precursor symptom. Despite initial treatment for eczema, the condition worsened over 10 months, leading to erythema, edema, and enlarged lymph nodes. Laboratory and imaging findings confirmed the diagnosis of MF. The patient responded partially to cyclophosphamide/doxorubicin/prednisone in combination with brentuximab vedotin (A-CHP) therapy. This case highlights the significance of recognizing blistering as a prodromal symptom for early detection and management of MF.

Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester.

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C(16) and C(18) unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.

Congenital Langerhans Cell Histiocytosis With the Skin and Lung Involvement: A Case and Literature Review.

Langerhans cell histiocytosis (LCH) is a clonal proliferative disease of immature Langerhans cells that expand in various organs, leading to organ and tissue dysfunction. Although LCH is most commonly seen in children under the age of three, a small number of cases of congenital LCH have been described. With a review of the literature on congenital LCH with lung and skin lesions, we present a case of congenital LCH with involvement of skin and lung, which was effectively treated with chemotherapy without recurrence for 3 years during the observational period. In addition, we summarized previously published case studies of congenital LCH with skin and lung involvement.

ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta.

Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.

Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea.

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in Synechococcus sp. PCC 7002.

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.