Cell Wall Glycosidases Research Articles

Candida albicans Mannosidases, Dfg5 and Dcw1, Are Required for Cell Wall Integrity and Pathogenesis.

Candida albicans is an oral mucosal commensal fungus that transforms into an opportunistic pathogen under specific conditions, including immunosuppression. It causes oral and systemic candidiasis, which results in a significant health burden. Furthermore, an alarming rise in antifungal drug resistance in Candida species raises the urgent need for novel drugs and drug targets. C. albicans Dfg5 and Dcw1 are homologous cell wall alpha-1,6-mannosidases with critical functions and represent potential new drug targets. Our past studies have shown that Dfg5 and Dcw1 function in cell wall biogenesis through the cross-linking of glycoproteins into the cell wall, thus playing a key role in cell wall integrity. Additionally, Dfg5 and Dcw1 are required for hyphal morphogenesis. However, the exact functions of Dfg5 and Dcw1 in cell wall integrity, hyphal morphogenesis, and pathogenesis are not known. In this study, we determined the relation of Dfg5 and Dcw1 with Hog1 MAPK, which plays a key role in cell wall integrity via the regulation of chitin synthesis in C. albicans. Additionally, we also determined the effects of dfg5 and dcw1 mutations on the gene expression of transcriptional regulators of hyphal morphogenesis. Furthermore, we determined the effects of dfg5 and dcw1 mutations on pathogenesis in a mouse model of oral candidiasis. Our results demonstrate that dfg5 and dcw1 mutations, as well as a hog1 knockout mutation, result in the dysregulation of chitin synthesis, resulting in a cell separation defect. Heterozygous and conditional mutations in dfg5 and dcw1 resulted in decreased transcriptional levels of cst20, a positive regulator of hyphal morphogenesis. However, dfg5 and dcw1 mutations resulted in increased levels of all the five negative regulators of hyphal morphogenesis-Tup1, Nrg1, Mig1, Rbf1, and Rfg1. Additionally, Tup1 levels were significantly higher than other negative regulators, indicating that Dfg5 and Dcw1 function in hyphal morphogenesis by repressing Tup1. Finally, dfg5 and dcw1 mutations affected the ability of C. albicans to cause oral candidiasis in mice. Thus, the cell wall glycosidases Dfg5 and Dcw1 are required for virulence and pathogenesis and represent novel drug targets.

Cell-Wall Hydrolases as Antimicrobials against Staphylococcus Species: Focus on Sle1.

Some staphylococcal species are opportunistic pathogens of humans and/or animals with Staphylococcus epidermidis as one of the most important. It causes a broad spectrum of diseases in humans and animals. This species is able to form biofilms and has developed antibiotic resistance, which has motivated research on new antibacterial agents. Cell-wall hydrolases (CWHs) can constitute a potential alternative. Following a hijacking strategy, we inventoried the CWHs of S. epidermidis. The lytic potential of representative CWHs that could be turned against staphylococci was explored by turbidity assays which revealed that cell wall glycosidases were not efficient, while cell wall amidases and cell wall peptidases were able to lyse S. epidermidis. Sle1, which is encoded by chromosomal gene and composed of three anchoring LysM domains and a C-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain, was one of the most active CWHs. The phylogeny of Sle1 revealed seven clusters mostly identified among staphylococci. Sle1 was able to lyse several staphylococcal species, including Staphylococcus aureus, both in planktonic and sessile forms, but not Micrococcus.

Cell Wall Hydrolases in Bacteria: Insight on the Diversity of Cell Wall Amidases, Glycosidases and Peptidases Toward Peptidoglycan.

The cell wall (CW) of bacteria is an intricate arrangement of macromolecules, at least constituted of peptidoglycan (PG) but also of (lipo)teichoic acids, various polysaccharides, polyglutamate and/or proteins. During bacterial growth and division, there is a constant balance between CW degradation and biosynthesis. The CW is remodeled by bacterial hydrolases, whose activities are carefully regulated to maintain cell integrity or lead to bacterial death. Each cell wall hydrolase (CWH) has a specific role regarding the PG: (i) cell wall amidase (CWA) cleaves the amide bond between N-acetylmuramic acid and L-alanine residue at the N-terminal of the stem peptide, (ii) cell wall glycosidase (CWG) catalyses the hydrolysis of the glycosidic linkages, whereas (iii) cell wall peptidase (CWP) cleaves amide bonds between amino acids within the PG chain. After an exhaustive overview of all known conserved catalytic domains responsible for CWA, CWG, and CWP activities, this review stresses that the CWHs frequently display a modular architecture combining multiple and/or different catalytic domains, including some lytic transglycosylases as well as CW binding domains. From there, direct physiological and collateral roles of CWHs in bacterial cells are further discussed.

The structure of Resuscitation promoting factor B from M. tuberculosis reveals unexpected ubiquitin-like domains

BackgroundRpfB is a key factor in resuscitation from dormancy of Mycobacterium tuberculosis. This protein is a cell-wall glycosidase, which cleaves cell-wall peptidoglycan. RpfB is structurally complex and is composed of three types of domains, including a catalytic, a G5 and three DUF348 domains. Structural information is currently limited to a portion of the protein including only the catalytic and G5 domains. To gain insights into the structure and function of all domains we have undertaken structural investigations on a large protein fragment containing all three types of domains that constitute RpfB (RpfB3D). MethodsThe structural features of RpfB3D have been investigated combining x-ray crystallography and biophysical studies. Results and conclusionsThe crystal structure of RpfB3D provides the first structural characterization of a DUF348 domain and revealed an unexpected structural relationship with ubiquitin. The crystal structure also provides specific structural features of these domains explaining their frequent association with G5 domains. General significanceResults provided novel insights into the mechanism of peptidoglycan degradation necessary to the resuscitation of M. tuberculosis. Features of the DUF348 domain add structural data to a large set of proteins embedding this domain. Based on its structural similarity to ubiquitin and frequent association to the G5 domain, we propose to name this domain as G5-linked-Ubiquitin-like domain, UBLG5.

Expression of UME6 , a Key Regulator of Candida albicans Hyphal Development, Enhances Biofilm Formation via Hgc1- and Sun41-Dependent Mechanisms

Biofilm formation is associated with the ability of Candida albicans, the major human fungal pathogen, to resist antifungal therapies and grow on tissues, catheters, and medical devices. In order to better understand the relationship between C. albicans morphology and biofilm formation, we examined biofilms generated in response to expression of UME6, a key filament-specific transcriptional regulator. As UME6 levels rise, C. albicans cells are known to transition from yeast to hyphae, and we also observed a corresponding increase in the level of biofilm formation in vitro. In addition to forming a biofilm, we observed that a C. albicans strain expressing constitutive high levels of UME6 promoted tissue invasion in a reconstituted human three-dimensional model of oropharyngeal candidiasis. Confocal microscopy indicated that both the top and bottom layers of the biofilm generated upon high-level constitutive UME6 expression consist primarily of hyphal cells. UME6-driven biofilm formation was reduced upon deletion of Hgc1, a cyclin-related protein important for hyphal development, as well as Sun41, a putative cell wall glycosidase. Constitutive high-level UME6 expression was also able to completely bypass both the filamentation and biofilm defects of a strain deleted for Efg1, a key transcriptional regulator of these processes. Finally, we show that both Sun41 and Efg1 affect the ability of UME6 to induce certain filament-specific transcripts. Overall, these findings indicate a strong correlation between increased C. albicans hyphal growth and enhanced biofilm formation and also suggest functional relationships between UME6 and other regulators of biofilm development.

Cell wall glycosidase activities and protein content variations during fruit development and ripening in three texture contrasted tomato cultivars

Excessive softening is the main factor limiting fruit shelf life and storage. It is generally acceptable now that softening of fruit which occurs during the ripening is due to synergistic actions of several enzymes on cell wall polysaccharides. As a subject for this study, we have assayed some glycosidase activities using three tomato species (Lycopersicon esculentum) contrasted for their texture phenotypes; the cherry tomato line Cervil (Solanum lycopersicum var. cerasiforme), a common taste tomato line Levovil (S. lycopersicum Mill.) and VilB a modern line, large, firmer and with good storage capability. Four glycosidase activities namely α-galactosidase, β-galactosidase, β-mannosidase and β-glucosidase were extracted from tomato’s cell wall of the three species. Cell wall protein from fruits pericarp was extracted and compared among the three cultivars at the following stages; 14days post anthesis (14DPA) fruit; 21days post anthesis (21DPA), turning (breaker), red and over ripe. When glycolytic activities were also compared among these cultivars at the precited development stages, gross variations were noticed from stage to stage and also from species to species in accordance with the fruit firmness status. Interestingly, VilB cultivar, the firmer among the other two, though possessed the highest total protein content, exhibited the lowest enzymatic activities. Taken together, these results may therefore allow us to conclude that studies of glycolytic activities in a single tomato cultivar cannot be generalized to all species. On the other hand, relating fruit development to glycosidase activities should logically be coupled to these enzymes from cell wall compartment.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a major fragment of the resuscitation-promoting factor RpfB fromMycobacterium tuberculosis

RpfB is required for the virulence and the resuscitation from dormancy of Mycobacterium tuberculosis, the bacterium responsible for tuberculosis. This protein is a cell-wall glycosidase that acts by cleaving peptidoglycans of the bacterial cell wall and therefore stimulates both bacterial growth and resuscitation from latency. RpfB consists of 362 residues organized into five domains. A long portion of RpfB, including its C-terminal catalytic domain, the G5 domain and one of its three DUF348 domains, which are of hitherto unknown structure and function, has been successfully crystallized using vapour-diffusion methods and seeding techniques. The crystals diffracted to 2.55 Å resolution and belonged to space group C222(1), with unit-cell parameters a=102.3, b=126.2, c=85.87 Å. Model building using phases derived from the combined use of multiwavelength anomalous dispersion and molecular replacement is in progress. The results obtained here will provide the first structural characterization of a DUF348 domain reported to date and will shed light on the functional role of the noncatalytic domains of RpfB.

Oligosaccharin and ABA synergistically affect the acquisition of freezing tolerance in winter wheat

In this paper, we continue our studies of the previously discovered [O.A. Zabotina, D.A. Ayupova, O.N. Larskaya, O.N. Nikolaeva, G.I. Petrovicheva, A.I. Zabotin, Physiologically active oligosaccharides, accumulating in the roots of winter wheat during adaptation to low temperature, Russian Journal of Plant Physiology 45 (1998) 262] oligosaccharin (physiologically active oligosaccharide) GXAG, which stimulates the acquisition of freezing tolerance in winter varieties of Triticum aestivum L. The transient accumulation of GXAG in the tissues of winter wheat correlates with the temporal activation of cell wall glycosidases during the first hours of cold acclimation (2 degrees C). This finding suggests that the oligosaccharin is liberated as a result of the intensification of hemicellulose turnover. At low concentrations, GXAG initiates the acquisition of freezing tolerance in winter plants, in a manner similar to ABA, even at room temperature. The resultant effect of ABA and GXAG on the freezing tolerance of winter wheat depends on the sequence of pre-treatments with these two factors. When seedlings are pre-treated with GXAG a few hours before treatment with ABA, the effect is synergistic, and its impact depends on the duration of pre-treatment with GXAG. When ABA is applied first, the resultant effect on freezing tolerance is additive. The results obtained here lead to the conclusion that oligosaccharin, accumulating during the first hours of cold acclimation, functions as a partner of ABA during the initiation of freezing tolerance acquisition in winter plants. We hypothesize that GXAG increases cell receptivity to ABA signaling.

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth

The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments ( Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (β- d-glucosidase, β- d-mannosidase, β- d-galactosidase, β- d-xylosidase, α- d-galactosidase, and α- l-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme α- d-galactosidase increased while the β- d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.

RBR1, a novel pH-regulated cell wall gene of Candida albicans, is repressed by RIM101 and activated by NRG1.

The transcription factor Rim101p of Candida albicans has been shown to play a major role in pH-dependent gene regulation. Rim101p is involved in cell wall biosynthesis, since it regulates PHR1 and PHR2, two almost functionally redundant cell wall glycosidases important for adaptation to either neutral or acidic habitats within the human host. To identify additional cell wall components regulated by Rim101p, we performed transcriptional profiling with a cell wall-specific DNA microarray. We showed that Rim101p contributes to the activation of known hypha-specific genes such as HWP1 and RBT1 but is also required for repression of the previously uncharacterized potential cell wall genes RBR1, RBR2, and RBR3. Further characterization of RBR1 revealed that it encodes a small glycosylphosphatidyl inositol protein that is expressed under acidic conditions predominantly at low temperature. Deletion of the gene resulted in a filamentation defect at low pH. Most interestingly, NRG1, a transcriptional repressor of hyphal growth in C. albicans, was required for RBR1 expression. The apparently activating effect of NRG1 observed in this study has not been described before. In addition, we showed that expression of NRG1 is not only temperature but also pH dependent.

Evidence Against the Involvement of Galactosidase or Glucosidase in Auxin- or Acid-stimulated Growth

Research on the mode of action of auxin in the promotion of growth has shown that auxin treatment leads to hydrogen ion secretion and wall acidification. It has recently been reported that auxin stimulates cell wall beta-galactosidase activity in Avena coleoptiles, presumably by causing cell wall acidification, since the pH optimum for the enzyme is about 5.0. It has been suggested that enhancement of beta-galactosidase and/or other glycosidase activity mediates growth promotion by auxin or low pH. This hypothesis was tested by examining the effect of inhibitors of beta-galactosidase and beta-glucosidase. Severe inhibition of measureable beta-galactosidase or beta-glucosidase activity was found to have no effect on auxin- or acid-promoted growth. It is concluded that neither beta-galactosidase nor beta-glucosidase plays an important role in short term growth promotion by auxin or acid. The data do not rule out the possibility that some other cell wall glycosidase is involved in auxin or acid action.

Activation of Avena Coleoptile Cell Wall Glycosidases by Hydrogen Ions and Auxin

Several cell wall-bound glycosidases present in Avena sativa coleoptiles were assayed by following the hydrolysis of p-nitrophenyl-glycosides. Particular emphasis was placed on characterizing some parameters affecting the activity of beta-galactosidase. The pH optimum of this enzyme is 4.5 to 5.5; it is sensitive to copper ions and p-chloromercuribenzoate treatment and apparently has an exceptionally low turnover rate. Indoleacetic acid treatment enhanced in vivo beta-galactosidase activity of coleoptile segments by 36% over control after 60 minutes. This enhancement was prevented by abscisic acid and cycloheximide. High buffer strengths and low pH reduced the indoleacetic acid-enhanced increase in enzyme activity. These data lend support to the following proposed model of indoleacetic acid action. Indoleacetic acid enhances the release of hydrogen ions into the cell wall which promote the activities of cell wall glycosidases, some of which may participate in the cell extension process.

Retention of a Gum Elastic Bougie for more than Eleven Months in the Uterus

ABSTRACT Biofilm formation is associated with the ability of Candida albicans, the major human fungal pathogen, to resist antifungal therapies and grow on tissues, catheters, and medical devices. In order to better understand the relationship between C. albicans morphology and biofilm formation, we examined biofilms generated in response to expression of UME6, a key filament-specific transcriptional regulator. As UME6 levels rise, C. albicans cells are known to transition from yeast to hyphae, and we also observed a corresponding increase in the level of biofilm formation in vitro. In addition to forming a biofilm, we observed that a C. albicans strain expressing constitutive high levels of UME6 promoted tissue invasion in a reconstituted human three-dimensional model of oropharyngeal candidiasis. Confocal microscopy indicated that both the top and bottom layers of the biofilm generated upon high-level constitutive UME6 expression consist primarily of hyphal cells. UME6-driven biofilm formation was reduced upon deletion of Hgc1, a cyclin-related protein important for hyphal development, as well as Sun41, a putative cell wall glycosidase. Constitutive high-level UME6 expression was also able to completely bypass both the filamentation and biofilm defects of a strain deleted for Efg1, a key transcriptional regulator of these processes. Finally, we show that both Sun41 and Efg1 affect the ability of UME6 to induce certain filament-specific transcripts. Overall, these findings indicate a strong correlation between increased C. albicans hyphal growth and enhanced biofilm formation and also suggest functional relationships between UME6 and other regulators of biofilm development.