Cell Wall Function Research Articles

BioSpotlight

BioSpotlight

The Role of Receptor-Like Kinases in Regulating Cell Wall Function

The Role of Receptor-Like Kinases in Regulating Cell Wall Function

Cinétique d’action du para-guanidinoéthylcalix[4]arène, et évolution de la perméabilité membranaire

Three problems at the moment: multidrug-resistant bacteria, healthcare-associated infections, and decrease of active antibiotics. We have an urgent need of new antibacterials, with an innovative mechanism of action, in order to avoid too quickly bacterial resistance. The first interface between bacteria and antibiotics is the bacterial cell wall. It is a very interesting target, as far as some components or motives are highly conserved between genus or species, and wall destabilization conduct rapidly to bacterial lysis. However, few methods are at our disposal to study rapidly impact of such antibacterials on the structure, composition or functions of the bacterial cell wall. The paraguanidinoethylcalix[4]arene (Cx1) is a new cationic antibacterial drug, with a broad spectrum, not toxic, active on multidrug-resistant bacteria, with a possible parietal target, but with unknown kind of activity (i.e. bactericidal or bacteriostatic). We thus developed, at the same time as the realization of the time-kill curves, a technique to stain bacteria with two dyes: SYTO9 ® and propidium iodide (PI), to follow the membrane permeability modifications, due to Cx1 exposure. The obtained results demonstrate, for Escherichia coli ATCC 25922, that Cx1 possesses a bactericidal activity, concentration-dependent, with a gradual achievement of membrane permeability, time- and concentration-dependent, with the presence of filamentous bacteria. In conclusion: the SYTO9 ®-PI double staining, allows a simple and fast detection, easy to implement, of the impact of new antibacterial on the bacterial wall; and Cx1 interacts well with the bacterial wall, pulling in the end a loss of membrane integrity.

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase-fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization. Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.

Interactions between salinity and boron toxicity in tomato plants involve apoplastic calcium

The lack of consensus about the mutual relations between salinity and boron (B) toxicity with respect to the physiological response of plants necessitates investigation of the interactions of soluble B with salinity. In this investigation, the effect of B was compared with Ca in order to elucidate whether the two nutrients have similar effects and/or to elucidate a relationship under salinity. Following addition of B or Ca, salinity was applied to tomato plants and the cell wall and plasma membrane permeability, measured as water permeability and electrolyte leakage, in relation to amino acid and ion cell wall composition, were determined. As the relationship between B and salinity was complex, several hypotheses are established. The increase of aquaporin functionality due to the presence of B and Ca compared with NaCl-treated plants could be the most feasible, whereas there is currently no satisfactory explanation for the results for the cell wall amino acid composition. In addition, the elemental composition results revealed that, in addition the known interactions between B and Ca with respect to cell wall stability, Mg and Mn were also increased in NaCl+B and NaCl+Ca treatments, suggesting their possible involvement in the cell wall function necessary for plant growth.

Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up ∼90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of α-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.

Proteomic analysis of hybrid poplar xylem sap

Xylem sap collected from Populus trichocarpa × Populus deltoides using root pressure was estimated to contain more than 100 proteins. Ninety-seven of these proteins were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). These proteins were classified into 10 functional categories including metabolism, signaling, stress response and cell wall functions. The majority of xylem sap proteins were metabolic enzymes involved in processes including translation, proteolysis, and glycolysis. Stress-related proteins were also prevalent. In contrast to xylem sap proteins collected from annual plants, the majority of poplar xylem sap proteins do not appear to be classically secreted since only 33 proteins were predicted to have an N-terminal signal peptide targeting them to the secretory pathway. Of the remaining 64 proteins, 27 were predicted to be secreted non-classically. While a number of proteins identified here have been previously reported in xylem sap proteomes of annual plants, many xylem sap proteins were identified in poplar which may reflect functions specific to perennial plants.

Analysis of Cell Membrane Characteristics of In Vitro-Selected Daptomycin-Resistant Strains of Methicillin-Resistant Staphylococcus aureus

Our previous studies of clinical daptomycin-resistant (Dap(r)) Staphylococcus aureus strains suggested that resistance is linked to the perturbations of several key cell membrane (CM) characteristics, including the CM order (fluidity), phospholipid content and asymmetry, and relative surface charge. In the present study, we examined the CM profiles of a well-known methicillin-resistant Staphylococcus aureus (MRSA) strain (MW2) after in vitro selection for DAP resistance by a 20-day serial passage in sublethal concentrations of DAP. Compared to levels for the parental strain, Dap(r) strains exhibited (i) decreased CM fluidity, (ii) the increased synthesis of total lysyl-phosphatidylglycerol (LPG), (iii) the increased flipping of LPG to the CM outer bilayer, and (iv) the increased expression of mprF, the gene responsible for the latter two phenotypes. In addition, we found that the expression of the dlt operon, which also increases positive surface charge, was enhanced in the Dap(r) mutants. These phenotypic and genotypic changes correlated with reduced DAP surface binding, mirroring observations made in clinical Dap(r) isolates. In this strain, serial exposure to DAP induced an increase in vancomycin MICs into the vancomycin-intermediate S. aureus (VISA) range (4 microg/ml) in parallel with increasing DAP MICs. Also, this Dap(r) strain exhibited significantly thicker cell walls than the parental strain, potentially correlating with the coevolution of the VISA phenotype and implicating cell wall structure and/or function in the Dap(r) phenotype. Importantly, despite the overexpression of mprF and dlt, the relative net positive surface charge was decreased in the Dap(r) mutants, suggesting that other factors contribute to the surface charge alterations and that a simple charge repulsion mechanism could not entirely explain the Dap(r) phenotype in these strains.

Exposure to caspofungin activates Cap and Hog pathways inCandida albicans

Caspofungin is a member of the echinocandin group of antifungals and inhibits the activity of beta-glucan synthase thus disrupting cell wall formation and function. While the potent antifungal activity of this agent is well established, this paper analyzed the response of Candida albicans to caspofungin. Exposure of yeast cells to 0.19 microg/ml caspofungin for 1 to 4 h induced nuclear translocation of Cap1p which was confirmed by Western blotting and confocal microscopy. Caspofungin-treated cells demonstrated increased expression of a number of genes associated with the oxidative stress response, including glutathione reductase (GLR1), mitochondrial processing protease (MAS1) and manganese-superoxide dismutase (SOD2) as well as elevated activity of glutathione reductase and superoxide dismutase. Caspofungin treatment also leads to the nuclear localization of Hog1p as visualized by Western blot using anti-phospho-p38 MAPK (Thr180/Tyr182) antibody. This translocation event lead to increased mRNA levels of catalase (CAT1) but not alkyl hydroperoxide reductase (AHP1). The activity of catalase was increased and reached a maximum at 2 h. In addition, pre-exposure of C. albicans to hydrogen peroxide (0.5 mM, 60 min) conferred an increased tolerance to caspofungin. The data presented here highlight the potent antifungal activity of caspofungin and demonstrate that upon exposure to this agent, C. albicans activates the Cap and Hog pathways in an attempt to limit the oxidative and osmotic stresses associated with this drug.

Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes

To further understand the roles of protein glycosylation in eukaryotes, we globally identified glycan-containing proteins in yeast. A fluorescent lectin binding assay was developed and used to screen protein microarrays containing over 5000 proteins purified from yeast. A total of 534 yeast proteins were identified that bound either Concanavalin A (ConA) or Wheat-Germ Agglutinin (WGA); 406 of them were novel. Among the novel glycoproteins, 45 were validated by mobility shift upon treatment with EndoH and PNGase F, thereby extending the number of validated yeast glycoproteins to 350. In addition to many components of the secretory pathway, we identified other types of proteins, such as transcription factors and mitochondrial proteins. To further explore the role of glycosylation in mitochondrial function, the localization of four mitochondrial proteins was examined in the presence and absence of tunicamycin, an inhibitor of N-linked protein glycosylation. For two proteins, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through the cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization.

Two Leucine-Rich Repeat Receptor Kinases Mediate Signaling, Linking Cell Wall Biosynthesis and ACC Synthase in Arabidopsis

The plant cell wall is a dynamic structure that changes in response to developmental and environmental cues through poorly understood signaling pathways. We identified two Leu-rich repeat receptor-like kinases in Arabidopsis thaliana that play a role in regulating cell wall function. Mutations in these FEI1 and FEI2 genes (named for the Chinese word for fat) disrupt anisotropic expansion and the synthesis of cell wall polymers and act additively with inhibitors or mutations disrupting cellulose biosynthesis. While FEI1 is an active protein kinase, a kinase-inactive version of FEI1 was able to fully complement the fei1 fei2 mutant. The expansion defect in fei1 fei2 roots was suppressed by inhibition of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, an enzyme that converts Ado-Met to ACC in ethylene biosynthesis, but not by disruption of the ethylene response pathway. Furthermore, the FEI proteins interact directly with ACC synthase. These results suggest that the FEI proteins define a novel signaling pathway that regulates cell wall function, likely via an ACC-mediated signal.

Equilibrium adsorption isotherm and controlled release of antibiotic drug chloroamphenicol from poly(2‐vinyl pyridine/acrylic acid) hydrogels prepared by gamma radiation

AbstractHigh water sorption of 2‐vinyl pyridine (2‐VP)/acrylic acid (AAc) hydrogel were prepared by free‐radical polymerization in aqueous solution of 2‐VP with AAc as comonomer. The amount of ionic monomer (AAc), the irradiation dose of prepared hydrogel, the pH, and the concentration of drug play an important factor on loading, adsorption, and releasing of water‐soluble chloroamphenicol drug. As a result of dynamic swilling tests, the effect of relative content of AAc on the swelling showed that it allowed a non‐Fickian type of water diffusion. The adsorption of the drug onto (2‐VP/AAc) hydrogels was studied by Freundlish adsorption isotherm. The drug concentrations showed an influence on the adsorption of drug which increased with increasing AAc content. From Freundlish adsorption isotherm, the empirical constants, k and n, can be evaluated and showed the ability of hydrogel to be loaded by the drug and the affinity of the drug to be uptake onto the hydrogel respectively. FTIR, TGA, and SEM techniques were used to study the characterization of hydrogel (2‐VP/AAc). Additionally, the release of the drug loaded from hydrogel discs was studied microbiologically to show that hydrophilic structure of the hydrogel has an antimicrobial effect as a dehydration of cytoplasm and unbalance of the cell wall functions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogensis, and conidiation

Growing resistance to current anti-fungal drugs is spurring investigation of new targets, including those in fungal wall metabolism. Galactofuranose (Gal f) is found in the cell walls of many fungi including Aspergillus fumigatus, which is currently the most prevalent opportunistic fungal pathogen in developed countries, and A. nidulans, a closely-related, tractable model system. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose into UDP-Gal f prior to incorporation into the fungal wall. We deleted the single-copy UGM sequence (AN3112.4, which we call ugmA) from an A. nidulans nkuAΔ strain, creating ugmAΔ. Haploid ugmAΔ strains were able to complete their asexual life cycle, showing that ugmA is not essential. However, ugmAΔ strains had compact colonial growth, which was associated with substantially delayed and abnormal conidiation. Compared to a wildtype morphology strain, ugmAΔ strains had aberrant hyphal morphology, producing wide, uneven, highly-branched hyphae, with thick, relatively electron-dense walls as visualized by transmission electron microscopy. These effects were partially remediated by growth on high osmolarity medium, or on medium containing 10 μg/mL Calcofluor, consistent with Gal f being important in cell wall structure and/or function.

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN-LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)(1).

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan-proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the β-d-glycosyl-Yariv (β-GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Epitopes of alkali-soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti-xyloglucan (anti-XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC-M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti-XG antibody at the trans-sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)-β-glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)-β-glucan (callose) could not be detected. The analytical results revealed that alkali-soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)-β-d-glucan.

Cell wall chitosaccharides are essential components and exposed patterns of the phytopathogenic oomycete Aphanomyces euteiches.

Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-D-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants.

Plant cell walls: New insights from ancient species

Cell walls are a defining feature of plants and have numerous crucial roles in growth and development. They are also the largest source of terrestrial biomass and have many important industrial applications – ranging from bulk products to functional food ingredients. There is considerable interest in the structure and functions of cell walls, and in the evolution of their remarkably complex polysaccharide structures. The grasses and cereals (order Poales), have long been regarded as being unique in that their cell walls contain an unbranched homopolymer, (1→3)(1→4)-β-D-glucan, in which short blocks of (1→4)-linked β-D-Glcp are joined by occasional (1→3)-linkages. This mixed linkage glucan (MLG) has been the subject of extensive research because of the economic importance of several Poales species including rice, barley and wheat and because MLG has proven health benefits. The recent discovery of MLG in horsetails (Equisetales order) was therefore significant and has prompted a re-evaluation of some of the current views on cell wall evolution and structural diversity.Addendum to: Sørensen I, Pettolino FA, Wilson SM, Doblin MS, Johansen B, Bacic A, Willats WGT. Mixed-linkage (1→3),(1→4)-β-D-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls. Plant J 2008; 54:510-21.

Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae

BackgroundThe Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. We previously used transcriptome profiling with DNA microarrays to identify 46 potential Zap1 target genes in the yeast genome. In this new study, we used complementary methods to identify additional Zap1 target genes.ResultsWith alternative growth conditions for the microarray experiments and a more sensitive motif identification algorithm, we identified 31 new potential targets of Zap1 activation. Moreover, an analysis of the response of Zap1 target genes to a range of zinc concentrations and to zinc withdrawal over time demonstrated that these genes respond differently to zinc deficiency. Some genes are induced under mild zinc deficiency and act as a first line of defense against this stress. First-line defense genes serve to maintain zinc homeostasis by increasing zinc uptake, and by mobilizing and conserving intracellular zinc pools. Other genes respond only to severe zinc limitation and act as a second line of defense. These second-line defense genes allow cells to adapt to conditions of zinc deficiency and include genes involved in maintaining secretory pathway and cell wall function, and stress responses.ConclusionWe have identified several new targets of Zap1-mediated regulation. Furthermore, our results indicate that through the differential regulation of its target genes, Zap1 prioritizes mechanisms of zinc homeostasis and adaptive responses to zinc deficiency.

Mutations in the RAM network confer resistance to the thiol oxidant 4,4′-dipyridyl disulfide

Thiol oxidants are expected to have multiple effects in living cells. Hence, mutations giving resistance to such agents are likely to reveal important targets and/or mechanisms influencing the cellular capacity to withstand thiol oxidation. A screen for mutants resistant to the thiol-specific oxidant dipyridyl disulfide (DPS) yielded tao3-516, which is impaired in the function of the RAM signaling network protein Tao3/Pag1p. We suggest that the DPS-resistance of the tao3-516 mutant might be due to deficient cell-cycle-regulated production of the chitinase Cts1p, which functions in post-mitotic cell separation and depends on Tao3p and the RAM network for regulated expression. Consistent with this, deletion of other RAM genes or CTS1 also resulted in increased resistance to DPS. Exposure to DPS caused extensive depolarization of the actin cytoskeleton. We found that tao3-516 is resistant to latrunculin, a specific inhibitor of actin polymerization, and that ram, Deltaace2, and Deltacts1 mutants are resistant to benomyl, a microtubule-destabilizing drug. Since septum build-up depends on the organization of cytoskeletal proteins, the resistance to cytoskeletal stress of Cts1p-deficient mutants might relate to bypass for abnormal septum-associated protein sorting. The broad resistance toward oxidants (DPS, diamide and H(2)O(2)) of the Deltacts1 strain links cell wall function to the resistance to oxidative stress and suggests the existence of targets that are common for these oxidants.

Aro Mutations in Salmonella enterica Cause Defects in Cell Wall and Outer Membrane Integrity

In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.