Cell Wall Receptors Research Articles

Candida albicans N-Linked Mannans Potentiate the Induction of Trained Immunity via Dectin-2.

The interaction between the Candida albicans cell wall and pattern recognition receptors is crucial for the initiation of host immune responses, which, ultimately, contribute to the clearance of this pathogenic fungus. In the present study, we investigate the ability of C. albicans mannans to modulate immune response and induce innate immune memory (also termed trained immunity). Using mutants of C. albicans that are defective in or lack mannosyl residues, we show that alterations in the mannosylation of the C. albicans cell wall affect the innate cytokine response and strongly reduce the secretion of T-cell-derived cytokines. Subsequently, we demonstrate that the branching of N-linked mannan, but not O-linked mannan, is essential to potentiate the induction of trained immunity, a process mediated by dectin 2. In conclusion, N-linked mannan is needed, in addition to β-glucans, for an effective induction of trained immunity by C. albicans.

Transcriptome-wide analysis of North-East Indian rice cultivars in response to Bipolaris oryzae infection revealed the importance of early response to the pathogen in suppressing the disease progression.

Brown spot disease (BSD) of rice (Oryza sativa L.) caused by Bipolaris oryzae is one of the major and neglected fungal diseases worldwide affecting rice production. Despite its significance, very limited knowledge on genetics and genomics of rice in response to B. oryzae available. Our study firstly identified moderately resistant (Gitesh) and susceptible (Shahsarang) North-East Indian rice cultivars in response to a native Bipolaris oryzae isolate BO1. Secondly, a systematic comparative RNA seq was performed for both cultivars at four different time points viz. 12, 24, 48, and 72 hours post infestation (hpi). Differential gene expression analysis revealed the importance of early response to the pathogen in suppressing disease progression. The pathogen negatively regulates the expression of photosynthetic-related genes at early stages in both cultivars. Of the cell wall modification enzymes, cellulose synthase and callose synthase are important for signal transduction and defense. Cell wall receptors OsLYP6, OsWAK80 might positively and OsWAK25 negatively regulate disease resistance. Jasmonic acid and/or abscisic acid signaling pathways are presumably involved in disease resistance, whereas salicylic acid pathway, and an ethylene response gene OsEBP-89 in promoting disease. Surprisingly, pathogenesis-related proteins showed no antimicrobial impact on the pathogen. Additionally, transcription factors OsWRKY62 and OsWRKY45 together might negatively regulate resistance to the pathogen. Taken together, our study has identified and provide key regulatory genes involved in response to B. oryzae which serve as potential resources for functional genetic analysis to develop genetic tolerance to BSD of rice.

The Cell Wall Integrity Receptor Mtl1 Contributes to Articulate Autophagic Responses When Glucose Availability Is Compromised.

Mtl1protein is a cell wall receptor belonging to the CWI pathway. Mtl1 function is related to glucose and oxidative stress signaling. In this report, we show data demonstrating that Mtl1 plays a critical role in the detection of a descent in glucose concentration, in order to activate bulk autophagy machinery as a response to nutrient deprivation and to maintain cell survival in starvation conditions. Autophagy is a tightly regulated mechanism involving several signaling pathways. The data here show that in Saccharomyces cerevisiae, Mtl1 signals glucose availability to either Ras2 or Sch9 proteins converging in Atg1 phosphorylation and autophagy induction. TORC1 complex function is not involved in autophagy induction during the diauxic shift when glucose is limited. In this context, the GCN2 gene is required to regulate autophagy activation upon amino acid starvation independent of the TORC1 complex. Mtl1 function is also involved in signaling the autophagic degradation of mitochondria during the stationary phase through both Ras2 and Sch9, in a manner dependent on either Atg33 and Atg11 proteins and independent of the Atg32 protein, the mitophagy receptor. All of the above suggest a pivotal signaling role for Mtl1 in maintaining correct cell homeostasis function in periods of glucose scarcity in budding yeast.

Micafungin-Induced Cell Wall Damage Stimulates Morphological Changes Consistent with Microcycle Conidiation in Aspergillus nidulans.

Fungal cell wall receptors relay messages about the state of the cell wall to the nucleus through the Cell Wall Integrity Signaling (CWIS) pathway. The ultimate role of the CWIS pathway is to coordinate repair of cell wall damage and to restore normal hyphal growth. Echinocandins such as micafungin represent a class of antifungals that trigger cell wall damage by affecting synthesis of β-glucans. To obtain a better understanding of the dynamics of the CWIS response and its multiple effects, we have coupled dynamic transcriptome analysis with morphological studies of Aspergillus nidulans hyphae in responds to micafungin. Our results reveal that expression of the master regulator of asexual development, BrlA, is induced by micafungin exposure. Further study showed that micafungin elicits morphological changes consistent with microcycle conidiation and that this effect is abolished in the absence of MpkA. Our results suggest that microcycle conidiation may be a general response to cell wall perturbation which in some cases would enable fungi to tolerate or survive otherwise lethal damage.

In silico prediction and molecular docking study on the interaction of bioactive compounds of Adenanthera pavonina exploring the potential antifungal activity against Candida glabrata cell wall proteins

Candida glabrata infections being resistant to many azole antifungal agents, are difficult to treat. Various parts of Adenanthera pavonina have been used in traditional medicine. In the present study, an attempt was made to screen the bioefficacy of the identified phytoconstituents of A. pavonina on an in silico platform and identify some potential drug-like molecules that can impede important drug targets of C. glabrata using the molecular docking method. In a previous study related to the current research, the phytochemical profiling of the methanolic stem extract of A. pavonina was carried out using GC-MS to identify the phytoconstituents. The three-dimensional structure of the fungal receptors were derived by homology modeling using Modeller9v7 and the same for the ligands for which the structures were not available were drawn by ACD chemSketch. The docking of ligands and receptors were performed using PatchDock software. Druglikeliness and pharmacodynamics properties were evaluated using SWISS-ADME. GC-MS analysis of the A. pavonina extract revealed the presence of 17 phyto compounds, of which 2 heptyl 1,3dioxolane and methyl 4-o-methyl-d-arabinopyranoside best docked with the epithelial adhesion protein 6 receptor and cell wall transcription factor ACE2. Methyl 4-o-methyl-d-arabinopyranoside also best docked with the integral cell wall protein receptor. Although other compounds have shown good scores related to docking 2 heptyl 1, 3 dioxolane had an excellent binding affinity than the other ligands thus signifying its potent antifungal activity. 2 Heptyl 1, 3 Dioxolane was found to be BBB positive and 4-O-Methyl-D-arabinopyranoside is BBB negative. As the finding indicates, the two phyto compounds present in the methanolic stem extract of A. pavonina demonstrated good docking scores when docked with specific fungal cell wall receptors and thus can prove to be appropriated for the lead molecule.

Purification of a killer toxin from Aureobasidium pullulans for the biocontrol of phytopathogens.

The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, β-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including β-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii.

Molecular detection of G1 marker for orange rust resistance and analysis of candidate resistance <italic>WAK</italic> gene in sugarcane

<p indent=0mm>Sugarcane orange rust is an important fungal disease caused by <italic>Puccinia kuehnii</italic> Butler, which could lead to a reduction in sugarcane production and sugar content and cause serious losses to the sugarcane industry in worldwide. In this study, the molecular marker G1 was used to detect orange rust resistance genes in cultivars, ancestral species and the related <italic>genus</italic> of <italic>Saccharum </italic>in the world. The representative amplified bands were cloned, sequenced, functionally annotated, and clustered, and the origin and evolution of resistance genes was then analyzed. The results showed that 83 and 34 were detected with G1 marker, accounting for 66.9% and 67.4% in 124 Chinese and 46 foreign sugarcane cultivars, respectively. Among 34 sugarcane ancestral species and the related genus of <italic>Saccharum</italic>, 17 were detected by G1 marker, accounting for 50%, of which the highest percentage (100%) was in <italic>Saccharum spontaneum</italic>. Functional annotation revealed that G1 target gene encoded a wall-associated receptor-like kinase (WAK), and three proteins with high similarity were identified from the haploid proteome database of sugarcane cultivars. These proteins all contain the extracellular domain, transmembrane domain and intracellular domain with kinase activity of typical cell wall receptor structures. Phylogenetic analysis of nucleotide sequences clearly showed the origin and evolution of the candidate resistance <italic>WAK</italic> genes. Specifically, the <italic>WAK</italic> genes amplified by G1 marker could be divided into three groups. The first group is from <italic>S</italic>.<italic> spontaneum</italic> and <italic>Saccharum robustum</italic>. The second group is from <italic>S</italic>.<italic> robustum</italic>,<italic> Saccharum officinarum</italic> and <italic>Narenga porphyrooma</italic>. The third group is from <italic>S</italic>.<italic> spontaneum</italic>,<italic> S</italic>.<italic> robustum</italic>,<italic> Saccharum sinense</italic> and cultivars. These results provided an important support for breeding of sugarcane cultivars resistant to orange rust, and lay a foundation for further analysis of molecular mechanism of resistance genes.

New approach for the simultaneous detection of somatic coliphages and F-specific RNA coliphages as indicators of fecal pollution

Two groups of coliphages have been recently included in different water management policies as indicators of viral fecal pollution in water and food: somatic coliphages, which infect E. coli through cell wall receptors, and F-specific RNA coliphages, which infect through the F-pili. Somatic coliphages are more abundant in fecally contaminated waters, except reclaimed waters, those disinfected by UV irradiation, and some groundwater samples that show a higher level of F-specific coliphages. Somatic coliphages are morphologically similar to DNA enteric viruses while F-specific coliphages are similar to RNA viruses such as norovirus and hepatitis A viruses, which are the viral pathogens of concern in sewage. The use of strains sensitive to both types of phages has been proposed for total coliphage enumeration, thereby avoiding double analysis. The standardized methods available for coliphage detection are robust and cost-effective, but the introduction of ready-to-use methods would facilitate routine implementation in laboratories. The fastest available tool for somatic coliphage enumeration is the recently developed Bluephage, which uses a modified β-glucuronide-overexpressing E. coli strain unable to take up the glucuronide substrate. The overexpressed enzyme accumulates inside the bacterial cells until released by phage-induced cell lysis, whereupon it encounters its substrate and the medium changes from yellow to blue. The present method uses E. coli strain CB12, sensitive to somatic coliphages and F-specific coliphages due to the expression of the F-pili. The Bluephage approach incorporating CB12 detects both types of coliphages in a time range of 1:30 to 4:00 h, as assayed with coliphages from raw sewage, river water, sludge and mussels. This strategy can be applied to obtain qualitative and quantitative results and is applicable to microplates as well as to large sample volumes (100 ml). Moreover it can provide monitoring of water bodies at real time, as for example for ambient recreational beach monitoring.

Molecular recognition of lipopolysaccharide by the lantibiotic nisin

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli.

Purification and molecular characterization of a Metschnikowia saccharicola killer toxin lethal to a crab pathogenic yeast.

The marine yeast strain Metschnikowia saccharicola DD21-2, isolated from sediments in the Yalu River, produces a killer toxin with a lethal effect on Metschnikowia bicuspidate strain WCY, a pathogenic yeast strain that infects crabs. In this study, the killer toxin was purified and characterized. After sequential purification, the purity of the killer toxin was increased 72.2-fold over the purity of the yeast cell culture supernatant. The molecular weight of the purified killer toxin was 47.0 kDa. The optimal pH and temperature for killing activity were 5.5°C and 16°C, respectively. The killing activity was stable over a pH range of 4.0-6.5 and temperature range of 0°C-40°C. The purified killer toxin was only effective against toxin-sensitive integral cells and had no killing effect on the protoplasts of toxin-sensitive cells. When exerting the killing effect, the toxin bind to a cell wall receptor of the treated strain, disrupted cell wall integrity and eventually caused death. The amino acid sequence identified by mass spectroscopy indicated that the purified killer toxin might be a protein kinase, but did not show β-1,3-glucanase activity, consistent with the laminarin hydrolysis results. These findings provide a basis for disease prevention and control in marine aquaculture.

Deciphering how Cpl-7 cell wall-binding repeats recognize the bacterial peptidoglycan

Endolysins, the cell wall lytic enzymes encoded by bacteriophages to release the phage progeny, are among the top alternatives to fight against multiresistant pathogenic bacteria; one of the current biggest challenges to global health. Their narrow range of susceptible bacteria relies, primarily, on targeting specific cell-wall receptors through specialized modules. The cell wall-binding domain of Cpl-7 endolysin, made of three CW_7 repeats, accounts for its extended-range of substrates. Using as model system the cell wall-binding domain of Cpl-7, here we describe the molecular basis for the bacterial cell wall recognition by the CW_7 motif, which is widely represented in sequences of cell wall hydrolases. We report the crystal and solution structure of the full-length domain, identify N-acetyl-D-glucosaminyl-(β1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) as the peptidoglycan (PG) target recognized by the CW_7 motifs, and characterize feasible GMDP-CW_7 contacts. Our data suggest that Cpl-7 cell wall-binding domain might simultaneously bind to three PG chains, and also highlight the potential use of CW_7-containing lysins as novel anti-infectives.

The Role of Carbohydrates in the Lipopolysaccharide (LPS)/Toll-Like Receptor 4 (TLR4) Signalling.

The interactions between sugar-containing molecules from the bacteria cell wall and pattern recognition receptors (PRR) on the plasma membrane or cytosol of specialized host cells are the first molecular events required for the activation of higher animal’s immune response and inflammation. This review focuses on the role of carbohydrates of bacterial endotoxin (lipopolysaccharide, LPS, lipooligosaccharide, LOS, and lipid A), in the interaction with the host Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex. The lipid chains and the phosphorylated disaccharide core of lipid A moiety are responsible for the TLR4 agonist action of LPS, and the specific interaction between MD-2, TLR4, and lipid A are key to the formation of the activated complex (TLR4/MD-2/LPS)2, which starts intracellular signalling leading to nuclear factors activation and to production of inflammatory cytokines. Subtle chemical variations in the lipid and sugar parts of lipid A cause dramatic changes in endotoxin activity and are also responsible for the switch from TLR4 agonism to antagonism. While the lipid A pharmacophore has been studied in detail and its structure-activity relationship is known, the contribution of core saccharides 3-deoxy-d-manno-octulosonic acid (Kdo) and heptosyl-2-keto-3-deoxy-octulosonate (Hep) to TLR4/MD-2 binding and activation by LPS and LOS has been investigated less extensively. This review focuses on the role of lipid A, but also of Kdo and Hep sugars in LPS/TLR4 signalling.

Damage-associated responses of the host contribute to defence against cyst nematodes but not root-knot nematodes.

When nematodes invade and subsequently migrate within plant roots, they generate cell wall fragments (in the form of oligogalacturonides; OGs) that can act as damage-associated molecular patterns and activate host defence responses. However, the molecular mechanisms mediating damage responses in plant-nematode interactions remain unexplored. Here, we characterized the role of a group of cell wall receptor proteins in Arabidopsis, designated as polygalacturonase-inhibiting proteins (PGIPs), during infection with the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. PGIPs are encoded by a family of two genes in Arabidopsis, and are involved in the formation of active OG elicitors. Our results show that PGIP gene expression is strongly induced in response to cyst nematode invasion of roots. Analyses of loss-of-function mutants and overexpression lines revealed that PGIP1 expression attenuates infection of host roots by cyst nematodes, but not root-knot nematodes. The PGIP1-mediated attenuation of cyst nematode infection involves the activation of plant camalexin and indole-glucosinolate pathways. These combined results provide new insights into the molecular mechanisms underlying plant damage perception and response pathways during infection by cyst and root-knot nematodes, and establishes the function of PGIP in plant resistance to cyst nematodes.

Flocculation in industrial strains ofSaccharomyces cerevisiae: role of cell wall polysaccharides and lectin-like receptors

Yeast flocculation is the reversible aggregation of yeast cells promoted by the interaction between lectin-like protein receptors with mannose side chains on adjacent cell walls. Flocculation is governed by several physiological factors, including the type of nutrient sugar available to yeast. We grew four industrial strains of Saccharomyces cerevisiae, representing applications in the brewing, winemaking and bioethanol sectors, to late stationary phase and quantified the cellular content of mannans, glucans and lectin-like proteins on yeast cell surfaces. Results indicated that brewing and champagne strains showed moderate to high flocculation ability when grown with glucose, fructose, maltose or galactose, whereas winemaking and fuel alcohol strains only showed moderate flocculation when grown on maltose and galactose. All yeast strains studied were weakly flocculent when grown on mannose. With regard to lectin-like receptors, their number played a more important role in governing yeast flocculation than the mannan and glucan contents in yeast cell walls. We conclude that all the industrial strains of S. cerevisiae belonged to New-Flo type on the basis of their flocculation behaviour observed when cultured on different sugars. Quantification of yeast cell wall polysaccharides and receptor sites indicates that mannan and glucan levels remain almost constant, irrespective of the strain under investigation. The main difference in flocculation characteristics in industrial yeast strains appears to be due to variations in concentrations of lectin-like cell surface receptors. Our findings may benefit brewers, winemakers and other yeast-based technologies in design of media to prevent premature flocculation during fermentation. Copyright © 2017 The Institute of Brewing & Distilling

Transcriptional analysis of defense mechanisms in upland tetraploid switchgrass to greenbugs

BackgroundAphid infestation of switchgrass (Panicum virgatum) has the potential to reduce yields and biomass quality. Although switchgrass-greenbug (Schizaphis graminum; GB) interactions have been studied at the whole plant level, little information is available on plant defense responses at the molecular level.ResultsThe global transcriptomic response of switchgrass cv Summer to GB was monitored by RNA-Seq in infested and control (uninfested) plants harvested at 5, 10, and 15 days after infestation (DAI). Differentially expressed genes (DEGs) in infested plants were analyzed relative to control uninfested plants at each time point. DEGs in GB-infested plants induced by 5-DAI included an upregulation of reactive burst oxidases and several cell wall receptors. Expression changes in genes linked to redox metabolism, cell wall structure, and hormone biosynthesis were also observed by 5-DAI. At 10-DAI, network analysis indicated a massive upregulation of defense-associated genes, including NAC, WRKY, and MYB classes of transcription factors and potential ancillary signaling molecules such as leucine aminopeptidases. Molecular evidence for loss of chloroplastic functions was also detected at this time point. Supporting these molecular changes, chlorophyll content was significantly decreased, and ROS levels were elevated in infested plants 10-DAI. Total peroxidase and laccase activities were elevated in infested plants at 10-DAI relative to control uninfested plants. The net result appeared to be a broad scale defensive response that led to an apparent reduction in C and N assimilation and a potential redirection of nutrients away from GB and towards the production of defensive compounds, such as pipecolic acid, chlorogenic acid, and trehalose by 10-DAI. By 15-DAI, evidence of recovery in primary metabolism was noted based on transcript abundances for genes associated with carbon, nitrogen, and nutrient assimilation.ConclusionsExtensive remodeling of the plant transcriptome and the production of ROS and several defensive metabolites in an upland switchgrass cultivar were observed in response to GB feeding. The early loss and apparent recovery in primary metabolism by 15-DAI would suggest that these transcriptional changes in later stages of GB infestation could underlie the recovery response categorized for this switchgrass cultivar. These results can be exploited to develop switchgrass lines with more durable resistance to GB and potentially other aphids.

Meta-QTL for resistance to white mold in common bean.

White mold, caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary, is a major disease that limits common bean production and quality worldwide. The host-pathogen interaction is complex, with partial resistance in the host inherited as a quantitative trait with low to moderate heritability. Our objective was to identify meta-QTL conditioning partial resistance to white mold from individual QTL identified across multiple populations and environments. The physical positions for 37 individual QTL were identified across 14 recombinant inbred bi-parental populations (six new, three re-genotyped, and five from the literature). A meta-QTL analysis of the 37 QTL was conducted using the genetic linkage map of Stampede x Red Hawk population as the reference. The 37 QTL condensed into 17 named loci (12 previously named and five new) of which nine were defined as meta-QTL WM1.1, WM2.2, WM3.1, WM5.4, WM6.2, WM7.1, WM7.4, WM7.5, and WM8.3. The nine meta-QTL had confidence intervals ranging from 0.65 to 9.41 Mb. Candidate genes shown to express under S. sclerotiorum infection in other studies, including cell wall receptor kinase, COI1, ethylene responsive transcription factor, peroxidase, and MYB transcription factor, were found within the confidence interval for five of the meta-QTL. The nine meta-QTL are recommended as potential targets for MAS for partial resistance to white mold in common bean.

Idiotypic Antifungal Vaccination: Immunoprotection by Antiidiotypic Antibiotic Antibodies.

As implied by the idiotypic network theory, the interaction between the functional epitope of a microbicidal molecule (X) and its specific cell-wall receptor (RX) on sensitive microorganisms may be imaged by the bond between the idiotype (Id) of a neutralizing monoclonal antibody (anti-X Ab) and its anti-idiotype (anti-Id) X-like Ab (anti-anti-X Ab). Consequently, anti-X Ab Id may mimic RX acting as a vaccine (idiotypic vaccination) for the elicitation of protective anti-Id Abs with antibiotic activity (antibiobodies).

Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling

Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

The effect of the physical properties of the substrate on the kinetics of cell adhesion and crawling studied by an axisymmetric diffusion-energy balance coupled model.

In this paper an analytical approach to study the effect of the substrate physical properties on the kinetics of adhesion and motility behavior of cells is presented. Cell adhesion is mediated by the binding of cell wall receptors and substrate's complementary ligands, and tight adhesion is accomplished by the recruitment of the cell wall binders to the adhesion zone. The binders' movement is modeled as their axisymmetric diffusion in the fluid-like cell membrane. In order to preserve the thermodynamic consistency, the energy balance for the cell-substrate interaction is imposed on the diffusion equation. Solving the axisymmetric diffusion-energy balance coupled equations, it turns out that the physical properties of the substrate (substrate's ligand spacing and stiffness) have considerable effects on the cell adhesion and motility kinetics. For a rigid substrate with uniform distribution of immobile ligands, the maximum ligand spacing which does not interrupt adhesion growth is found to be about 57 nm. It is also found that as a consequence of the reduction in the energy dissipation in the isolated adhesion system, cell adhesion is facilitated by increasing substrate's stiffness. Moreover, the directional movement of cells on a substrate with gradients in mechanical compliance is explored with an extension of the adhesion formulation. It is shown that cells tend to move from soft to stiff regions of the substrate, but their movement is decelerated as the stiffness of the substrate increases. These findings based on the proposed theoretical model are in excellent agreement with the previous experimental observations.

Yeast killer toxin-like candidacidal Ab6 antibodies elicited through the manipulation of the idiotypic cascade.

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by β-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to β-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble β-1,3-glucan, but not by pustulan, a β-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.