Cell Wall Polymers Research Articles

The Light-Controlled Release of 2-fluoro-l-fucose, an Inhibitor of the Root Cell Elongation, from a nitrobenzyl-caged Derivative.

Glycan metabolic engineering is a powerful tool for studying the glycosylation in living plant cells. The use of modified monosaccharides such as deoxy or fluorine-containing glycosides has been reported as a powerful pharmacological approach for studying the carbohydrate metabolism. 1,3,4-tri-O-acetyl-2-fluoro-l-fucose (2F-Fuc) is a potent inhibitor of the plant cell elongation. After feeding plant seedlings with 2F-Fuc, this monosaccharide derivative is deacetylated and converted by the endogenous metabolic machinery into the corresponding nucleotide-sugar, which then efficiently inhibits Golgi-localized fucosyltransferases. Among plant cell wall polymers, defects in the fucosylation of the pectic rhamnogalacturonan-II cause a decrease in RG-II dimerization, which in turn induce the arrest of the cell elongation. In order to perform the inhibition of the cell elongation process in a spatio-temporal manner, we synthesized a caged 3,4-di-O-acetyl-1-hydroxy-2-fluoro-l-fucose (1-OH-2F-Fuc) derivative carrying a photolabile ortho-nitrobenzyl alcohol function at the anomeric position: 3,4-di-O-acetyl-1-ortho-nitrobenzyl-2-fluoro-l-fucose (2F-Fuc-NB). The photorelease of the trapped 1-OH-2F-Fuc was performed under a 365 nm LED illumination. We demonstrated that the in planta elimination by photoexcitation of the photolabile group releases free 2F-Fuc in plant cells, which in turn inhibits in a dose-dependent manner and, reversibly, the root cell elongation.

Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

In the last decade it has become clear that enzymes in the "BAHD" family of acyl-CoA transferases play important roles in the addition of phenolic acids to form ester-linked moieties on cell wall polymers. We focus here on the addition of two such phenolics-the hydroxycinnamates, ferulate and p-coumarate-to two cell wall polymers, glucuronoarabinoxylan and to lignin. The resulting ester-linked feruloyl and p-coumaroyl moities are key features of the cell walls of grasses and other commelinid monocots. The capacity of ferulate to participate in radical oxidative coupling means that its addition to glucuronoarabinoxylan or to lignin has profound implications for the properties of the cell wall - allowing respectively oxidative crosslinking to glucuronoarabinoxylan chains or introducing ester bonds into lignin polymers. A subclade of ~10 BAHD genes in grasses is now known to (1) contain genes strongly implicated in addition of p-coumarate or ferulate to glucuronoarabinoxylan (2) encode enzymes that add p-coumarate or ferulate to lignin precursors. Here, we review the evidence for functions of these genes and the biotechnological applications of manipulating them, discuss our understanding of mechanisms involved, and highlight outstanding questions for future research.

Fluorogenic properties of 4-dimethylaminocinnamaldehyde (DMACA) enable high resolution imaging of cell-wall-bound proanthocyanidins in plant root tissues.

Proanthocyanidins (PAs) are polymeric phenolic compounds found in plants and used in many industrial applications. Despite strong evidence of herbivore and pathogen resistance-related properties of PAs, their in planta function is not fully understood. Determining the location and dynamics of PAs in plant tissues and cellular compartments is crucial to understand their mode of action. Such an approach requires microscopic localization with fluorescent dyes that specifically bind to PAs. Such dyes have hitherto been lacking. Here, we show that 4-dimethylaminocinnamaldehyde (DMACA) can be used as a PA-specific fluorescent dye that allows localization of PAs at high resolution in cell walls and inside cells using confocal microscopy, revealing features of previously unreported wall-bound PAs. We demonstrate several novel usages of DMACA as a fluorophore by taking advantage of its double staining compatibility with other fluorescent dyes. We illustrate the use of the dye alone and its co-localization with cell wall polymers in different Populus root tissues. The easy-to-use fluorescent staining method, together with its high photostability and compatibility with other fluorogenic dyes, makes DMACA a valuable tool for uncovering the biological function of PAs at a cellular level in plant tissues. DMACA can also be used in other plant tissues than roots, however care needs to be taken when tissues contain compounds that autofluoresce in the red spectral region which can be confounded with the PA-specific DMACA signal.

Small-scale OrganoCat processing to screen rapeseed straw for efficient lignocellulose fractionation

Agricultural residues such as rapeseed straw can be a valuable source of cellulose, sugars, and aromatic molecules like lignin. Understanding its composition is crucial in order to develop suitable processing technology for the production of biofuel or biochemicals from rapeseed straw. Here, we developed a small-scale OrganoCat system to screen multiple technical conditions and different samples at higher throughput and utilize this system to analyze straw samples from a set of 14 genetically different Brassica lines on their processability. Correlation analysis was performed to investigate the effects of cell wall polymer features on rapeseed biomass disintegration. At comparably mild reaction conditions, the differences in recalcitrance towards OrganoCat fractionation within the set were especially associated with parameters such as pectic polysaccharide content, acetylation, and hemicellulose composition. These findings can subsequently be used to optimize and scale up the pretreatment and fractionation of lignocellulose derived from rapeseed straw.

The unique sugar conversion and complex CAZyme system of Trichoderma brev T069 during solid-state fermentation of cassava peel

Cassava peel is a lignocellulose-based agricultural by-product that can be degraded into reducing sugars as a raw material for energy. However, chemical and enzymatic treatment processes of cell wall polymers are expensive. We found that the filamentous fungus Trichoderma brev T069 can grow in a cassava peel matrix without any additional nutrient and degrade macromolecular carbohydrates, producing high levels of glucose and valuable hydrolytic enzymes. Therefore, this paper investigated the utilization strategy of T. brev T069 on cassava peels by dynamically analyzing its growth and hydrolytic enzyme system during solid fermentation, degradation of cassava peel components, and changes in reducing sugars. Results showed that T. brev T069 uses a "borrow-return" method of reducing sugar conversion, first absorbing fructose for growth for 3 days and later releasing glucose (99.56 mg/g) and cellobiose (24.69 mg/g) via hydrolases and high levels of β-glucosidase (1196.05 IU/g). Scanning electron microscopy and Fourier transform infrared spectrometry revealed the compositional breakdown and decreased carbohydrate contents of the peels. Label-free proteomics identified 257 carbohydrate-active enzymes (CAZymes), and functional annotation indicated that hemicellulases, lignin-modifying enzymes, and pectinases were induced at early stages, whereas amylases and cellulases were induced at later stages. A protein–protein interaction network revealed that protein degradation-related proteases and α-mannosidases may be involved in the realignment of enzymatic degradation strategies. This study explored the hydrolytic enzymatic potential of the Trichoderma CAZyme system and provided a practical approach for the biotransformation of tropical agricultural wastes into value-added products.

Cell Wall Glycan Changes in Different Brachypodium Tissues Give Insights into Monocot Biomass

The annual temperate grass Brachypodium distachyon has become a model system for monocot biomass crops and for understanding lignocellulosic recalcitrance to employ better saccharification and fermentation approaches. It is a monocot plant used to study the grass cell walls that differ from the cell walls of dicot plants such as the eudicot model Arabidopsis. The B. distachyon cell wall is predominantly composed of cellulose, arabinoxylans, and mixed-linkage glucans, and it resembles the cell walls of other field grasses. It has a vascular bundle anatomy similar to C3 grasses. These features make Brachypodium an ideal model to study cell walls. Cell walls are composed of polymers with complex structures that vary between cell types and at different developmental stages. Antibodies that recognize specific cell wall components are currently one of the most effective and specific molecular probes to determine the location and distribution of polymers in plant cell walls in situ. Here, we investigated the glycan distribution in the cell walls of the root and leaf tissues of Brachypodium by employing cell-wall-directed antibodies against diverse glycan epitopes. There are distinct differences in the presence of the epitopes between the root and leaf tissues as well as in the cell type level, which gives insights into monocot biomass.

Insights from molecular dynamics simulations for interfacial effects between polylactic acid and wood cell wall constituents

• The interface structure between wood cell wall components and polymer is studied. • Molecular insights into the differences in the adhesion behavior of polymers on the surface of wood cell wall component. • Provides insights for the structural design of biomass composites. Knowledge of the interfacial structure from an atomic or molecular perspective helps to optimize the manufacturing of nanocomposite materials with bottom-up structural design and controls the performance more effectively. In this work, molecular dynamics simulation was used to study the composition, structure, and mechanism of interactions at the interface between wood cell wall constituents (cellulose, lignin, and hemicellulose) and polylactic acid (PLA). Results showed that the interfacial adhesion between the components and polymer was affected by the surface structure and the diffusion and aggregation of the polymer. Compared with the crystalline surface, the amorphous structure could achieve greater bonding energy and lower surface energy, and thus improving the dispersibility of fillers in the polymer matrix. An orderly arrangement and denser packing of the polymer were observed on the crystalline surface and the diffusion capacity of the polymer was reduced due to reduction in local free volume.

Seasonal biochemical changes in Betula tortuosa Ledeb. annual rings in Alpine forest-tundra of Kuznetsk Ala Tau Mountains

Woody vegetation growth conditions have marked effects on hemicellulose, cellulose and lignin formation. In this study, the climatic responses of these major cell wall polymers in Betula tortuosa Ledeb. were analyzed. 35 annual rings (1980–2015) of Betula tortuosa Ledeb. trees growing in the alpine forest-tundra of Kuznetsk Ala Tau were studied using Fourier-transform infrared (FTIR) spectroscopy and pyrolysis–gas chromatography–mass spectrometry (Py–GC/MS). Analysis of the correlation of the resulting spectra and Py–GC/MS values with mean air temperature and precipitation showed that the polymeric composition of Betula tortuosa Ledeb. was mainly determined by June–August climate. The major factor limiting the development of the “unique” cell wall polymer composition of Betula tortuosa Ledeb. found in alpine forest-tundra of Kuznetsk Ala Tau was a deficit of heat. At the end of the growing season, precipitation had a largely negative impact on polymer formation in Betula tortuosa Ledeb. cell walls. The authors believe that combining FTIR spectroscopy and Py–GC/MS is an effective approach to quantifying the consequences of current climate trends for Siberian forest ecosystems.

Transcriptomic Evidence Reveals Low Gelatinous Layer Biosynthesis in Neolamarckia cadamba after Gravistimulation.

Trees can control their shape and resist gravity by producing tension wood (TW), which is a special wood that results from trees being put under stress. TW is characterized by the presence of a gelatinous layer (G layer) and the differential distribution of cell wall polymers. In this study, we investigated whether or not gravistimulation in N. cadamba resulted in TW with an obvious G layer. The results revealed an absence of an obvious G layer in samples of the upper side of a leaning stem (UW), as well as an accumulation of cellulose and a decrease in lignin content. A negligible change in the content of these polymers was recorded and compared to untreated plant (NW) samples, revealing the presence of a G layer either in much lower concentrations or in a lignified form. A transcriptomic investigation demonstrated a higher expression of cell wall esterase- and hydrolase-related genes in the UW, suggesting an accumulation of noncellulosic sugars in the UW, similar to the spectroscopy results. Furthermore, several G-layer-specific genes were also downregulated, including fasciclin-like arabinogalactan proteins (FLA), beta-galactosidase (BGAL) and chitinase-like proteins (CTL). The gene coexpression network revealed a strong correlation between cell-wall-synthesis-related genes and G-layer-synthesis-specific genes, suggesting their probable antagonistic role during G layer formation. In brief, the G layer in N. cadamba was either synthesized in a very low amount or was lignified during an early stage of growth; further experimental validation is required to understand the exact mechanism and stage of G layer formation in N. cadamba during gravistimulation.

Effects of Nanosilver and Heat Treatment on the Pull-Off Strength of Sealer-Clear Finish in Solid Wood Species.

Pull-off strength is an important property of solid wood, influencing the quality of paints and finishes in the modern furniture industry, as well as in historical furniture and for preservation and restoration of heritage objects. The thermal modification and heat treatment of solid wood have been the most used commercial wood modification techniques over the past decades globally. The effects of heat treatment at two mild temperatures (145 and 185 °C) on the pull-off strength of three common solid wood species, i.e., common beech (Fagus sylvatica L.), black poplar (Populus nigra L.), and silver fir (Abies alba Mill.), were studied in the present research work. The specimens were coated with an unpigmented sealer-clear finish based on an organic solvent. The results demonstrated a positive correlation between the density and pull-off strength in the solid wood species. Heat treatment at 145 °C resulted in an increase in the pull-off strength in all three species, due to the formation of new bonds in the cell-wall polymers. Thermal degradation of the polymers at 185 °C weakened the positive effect of the formation of new bonds, resulting in a largely unchanged pull-off strength in comparison with the control specimens. Impregnation with a silver nano-suspension decreased the pull-off strength in beech specimens. It was concluded that density is the decisive factor in determining the pull-off strength, having a significant positive correlation (R-squared value of 0.89). Heat treatment at lower temperatures is recommended, to increase pull-off strength. Higher temperatures can have a decreasing effect on pull-off strength, due to the thermal degradation of cell-wall polymers.

Effects of Biochar and Nitrogen Application on Rice Biomass Saccharification, Bioethanol Yield and Cell Wall Polymers Features.

Rice is a major food crop that produces abundant biomass wastes for biofuels. To improve rice biomass and yield, nitrogen (N) fertilizer is excessively used, which is not eco-friendly. Alternatively, biochar (B) application is favored to improve rice biomass and yield under low chemical fertilizers. To minimize the reliance on N fertilizer, we applied four B levels (0, 10, 20, and 30 t B ha-1) combined with two N rates (low-135 and high-180 kg ha-1) to improve biomass yield. Results showed that compared to control, the combined B at 20-30 t ha-1 with low N application significantly improved plant dry matter and arabinose (Ara%), while decreasing cellulose crystallinity (Crl), degree of polymerization (DP), and the ratio of xylose/arabinose (Xyl/Ara), resulting in high hexoses (% cellulose) and bioethanol yield (% dry matter). We concluded that B coupled with N can alter cell wall polymer features in paddy rice resulting in high biomass saccharification and bioethanol production.

Quantitative enzymatic-mass spectrometric analysis of the chitinous polymers in fungal cell walls

Chitin is an essential structural component of complex and dynamic fungal cell walls. It may be converted by partial or full deacetylation to yield chitosan. Here, we describe a method to quantify N-acetyl d-glucosamine (GlcNAc, A) and d-glucosamine (GlcN, D) units and, thus, total amount and average fraction of acetylation (x̅ FA) of the chitinous polymers by complete enzyme hydrolysis of the polymers followed by mass spectrometric analyses of the monomers. First, the native polymers were isotopically N-acetylated, then enzymatically hydrolyzed to A and R (2H3N-acetyl-d-glucosamine – former D) monomers. Relative abundances of A and R units were used to calculate x̅ FA, and a double-isotopically labeled internal standard R* ([13C2,2H3] N-acetyl-d-glucosamine) monomer was used to calculate the absolute amounts of GlcNAc and GlcN units present in the fungal samples. The method was validated using known chitosan polymers and is suitable for both purified cell walls and whole mycelia.

Influence of climate seasonality on the accumulation of carbohydrates and cell wall polymers in organs of three tree species of a restinga forest

Influence of climate seasonality on the accumulation of carbohydrates and cell wall polymers in organs of three tree species of a restinga forest

Effect of low molecular weight melamine-urea-formaldehyde resin impregnation on poplar wood pore size distribution and water sorption

This study aimed to investigate the relationship between the properties of pore size distribution and water sorption in melamine-urea-formaldehyde (MUF) resin-impregnated wood by scanning electron microscopy (SEM), nitrogen adsorption (NAD) measurement, mercury intrusion porosimetry (MIP), water-soaking, and moisture adsorption over saturated salt solutions. The results showed that MUF resin not only filled the macropores and mesopores of the wood, such as those of vessels and wood fibers, but also penetrated the cell walls and replaced some hygroscopic groups of cell wall polymers, thus significantly reducing the water sorption. The porosity and cumulative pore volume of the RI140 and RII140 samples (impregnated with 10% and 20% MUF resin solution, respectively, with each dried at 140 ℃) were 19.25% and 1.20 mL·g−1, and 9.85% and 0.89 mL·g−1, respectively, which were lower than those obtained for the control group (CK). The specific surface area of RII140 was lower than that of CK. The pore size range of the CK and RII140 samples was 3.1–22.9 nm and 2.5–5.1 nm, respectively. The water absorption rate and equilibrium moisture content values for MUF resin-impregnated wood were inversely proportional to the drying temperatures and weight percent gain (WPG). The MUF resin leaching efficiency was inversely proportional to the drying temperature but proportional to the WPG.

Impact of miscanthus lignin and arabinoxylan on Portland cement

Miscanthus biomass can be used to produce lightweight concrete. However, cell wall polymers leached in the alkaline cementitious medium can disturb cement setting. This is the case for grass lignin and grass arabinoxylan due to their specific alkali solubility. The main objective of this paper was to study the impact of lignin and of arabinoxylan from miscanthus biomass on the hydration of Portland cement and by electrical conductivity. To this end, dioxan lignin (DL) and arabinoxylan (AX) were extracted from miscanthus by methods preserving the main structural specificities of the native polymers. These DL and AX fractions were added to Portland cement (1–5% w/w in cement) and their impact on the electrical conductivity of cement/water mixtures was time-monitored. The novelty of this study lies in using polymers structurally similar to those of miscanthus fibers rather than commercially available ones, such as kraft lignin (KL). The addition of DL or of KL to cement/water mixture differently affected the electrical conductivity, which is most likely assignable to the severe structural degradation of KL during kraft process. The conductivity curves suggested that cement hydration was substantially delayed when DL % in cement was 3% or more while lower values had no impact. The results support the hypothesis that the access of water to cement grains was impeded by the adsorption of ionized lignin entities at their surface. When co-added to the cement (1.6 wt% each), the DL and AX fraction delayed cement hydration more substantially than when the same amounts were separately added. This unexpected synergy suggests that the miscanthus lignin and arabinoxylan polymers form lignin-carbohydrate complexes efficiently adsorbed on cement grains.

Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction

BackgroundPlant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities.ResultsTo establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes.ConclusionsThe two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance.3Ck-Q-KzsX1uPWsLmUjZUvVideo

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.

Architecture of the dynamic fungal cell wall.

The fungal cell wall is essential for growth and survival, and is a key target for antifungal drugs and the immune system. The cell wall must be robust but flexible, protective and shielding yet porous to nutrients and membrane vesicles and receptive to exogenous signals. Most fungi have a common inner wall skeleton of chitin and β-glucans that functions as a flexible viscoelastic frame to which a more diverse set of outer cell wall polymers and glycosylated proteins are attached. Whereas the inner wall largely determines shape and strength, the outer wall confers properties of hydrophobicity, adhesiveness, and chemical and immunological heterogeneity. The spatial organization and dynamic regulation of the wall in response to prevailing growth conditions enable fungi to thrive within changing, diverse and often hostile environments. Understanding this architecture provides opportunities to develop diagnostics and drugs to combat life-threatening fungal infections.

Genome-edited rice deficient in two 4-COUMARATE:COENZYME A LIGASE genes displays diverse lignin alterations.

The 4-coumarate:coenzyme A ligase (4CL) is a key enzyme that contributes to channeling metabolic flux in the cinnamate/monolignol pathway, leading to the production of monolignols, p-hydroxycinnamates, and a flavonoid tricin, the major building blocks of lignin polymer in grass cell walls. Vascular plants often contain multiple 4CL genes; however, the contribution of each 4CL isoform to lignin biosynthesis remains unclear, especially in grasses. In this study, we characterized the functions of two rice (Oryza sativa L.) 4CL isoforms (Os4CL3 and Os4CL4) primarily by analyzing the cell wall chemical structures of rice mutants generated by CRISPR/Cas9-mediated targeted mutagenesis. A series of chemical and nuclear magnetic resonance analyses revealed that loss-of-function of Os4CL3 and Os4CL4 differently altered the composition of lignin polymer units. Loss of function of Os4CL3 induced marked reductions in the major guaiacyl and syringyl lignin units derived from both the conserved non-γ-p-coumaroylated and the grass-specific γ-p-coumaroylated monolignols, with more prominent reductions in guaiacyl units than in syringyl units. In contrast, the loss-of-function mutation to Os4CL4 primarily decreased the abundance of the non-γ-p-coumaroylated guaiacyl units. Loss-of-function of Os4CL4, but not of Os4CL3, reduced the grass-specific lignin-bound tricin units, indicating that Os4CL4 plays a key role not only in monolignol biosynthesis but also in the biosynthesis of tricin used for lignification. Further, the loss-of-function of Os4CL3 and Os4CL4 notably reduced cell-wall-bound ferulates, indicating their roles in cell wall feruloylation. Overall, this study demonstrates the overlapping but divergent roles of 4CL isoforms during the coordinated production of various lignin monomers.

P017 Echinocandin resistance mechanism in Candida tropicalis and Candida glabrata

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjective Candida tropicalis and Candida glabrata account for 41.6% and 7.08% of total Candidaemia cases in India. Echinocandins are the first-line treatment option for these infections. Resistance to Echinocandins is rare with Candida sp. However, in recent years, has been noted across many centers. We determined the mechanism of echinocandin resistance in C. tropicalis and C. glabrata.Methods C. tropicalis and C. glabrata isolated from Candidaemia patients over a period of 3 years (August 2016-July 2019), identified by MALDI-TOF-MS were used in this study. Antifungal susceptibility testing was done following CLSI broth microdilution reference method (M 27A). FKS1 gene was sequenced using species-specific primers for the presence of any mutation. To determine any changes in the cell wall chitin and glucan contents, expression fold changes of chitin synthase (CHS1, CHS2, and CHS3), and glucan synthase genes upon caspofungin treatment were determined using real-time qPCR. These findings were correlated with cell wall chitin and glucan content determined by flowcytometry.ResultsA total of 3558 Candida species were isolated from patients of all age groups at our hospital. C. tropicalis was the predominant agent (34%), while the prevalence of C. glabrata was 6%. A total of 17 (8.5%) C. glabrata and 3 (0.25%) C. tropicalis exhibited reduced susceptibility to echinocandins. All these isolates carried a wild-type FKS genes.In C. tropicalis, inducible expression of Chs1, Chs2 and Chs3 genes were comparable among susceptible and resistant isolates1.8 (0.4-2.8) vs. 2.5 (0.9-6.6), P = .247); [0.7 (0.3-1.8) vs. 0.7 (0.2-1.6), P = .793]; [1.3 (0.14-4.8) vs. 1.1(0.48-1.7), P = .522], respectively. In concordance with gene expression, there was no significant difference in cell wall chitin contents among resistant and susceptible [14.37 (6.5-24.8) vs 16.28 (6.0-24.7), P = .114] C. tropicalis isolates. In contrast in resistant isolates of C. glabrata, caspofungin treatment resulted in significantly higher induction of chitin synthase genes compared to susceptible isolates; Chs1 [2.34 (0.24-9.71) vs. 1.56 (0.55-4.5) (P = .007)], CHS2 [1.59 (0.33-8.0) vs. (2.3 (0.69-6.15), P = .0006], and CHS3 gene [3.8 (0.13-12.73) vs. 1.9 (0.56-7.16), P <.0001]. Flowcytometric data in terms of chitin content, correlated well with expression changes as staining index was significantly higher in resistant compared to susceptible isolates [320 (198-535) vs. 164 (5.34-254.10) P = .0001]. Glucan synthase expression was comparable in susceptible and resistant isolates of C. tropicalis [3.47 (1.57-7.63) vs. 4.41 (0.41-17.51), (P = .518)]. However, glucan synthase gene expression was found significantly increased in resistant C. glabrata isolates compared to susceptible isolates; 3.10 (1.02-16.45) vs. 1.61 (0.13-7.67), P <.001.ConclusionWe evaluated the role of cell wall components in echinocandin resistance in isolates with reduced susceptibility to echinocandins but lacking an FKS1 mutation. While chitin was induced at higher levels in C. glabrata, a similar finding was not observed in C. tropicalis. This warrants further studies to elucidate the role of fungal cell wall polymers in resistance.