Expression Of Cell Wall Research Articles

Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Chlorine Dioxide Treatment Modulates Ripening-Related Genes and Antioxidant System to Improve the Storability of Tomato

Chlorine dioxide (ClO2) is used to maintain quality and safety of fresh produce. However, ClO2 action mechanism in fresh produce is unknown. In this study, firstly, we evaluated the efficacy of ClO2 treatment on the quality, chilling injury, and calyx molding of tomatoes stored at two different temperatures. Then, ClO2 effect on the expression of cell wall- and ripening-related genes and on the activity of antioxidant enzymes was investigated. Tomatoes were treated with gaseous ClO2 for 15 min before transferring them to 13°C for 12 days and/or 4°C for 14 days, followed by 5 days at 20°C (shelf-life conditions). ClO2 treatment marginally reduced the rate of respiration but did not affect ethylene production at 13°C and 4°C storage or at shelf-life conditions. When stored at 13°C, treatment with ClO2 reduced the loss of firmness, with concomitant repression of pectin esterase 1, a cell wall-related gene. Additionally, at 13°C storage conditions, ClO2 treatment maintained tomato quality in terms of soluble solid content, titratable acidity, and color and was associated with the downregulation of the ripening-relatedethylene response factors B3/C1/E1 and the induction of antioxidant genes encoding catalase and ascorbate peroxidase. At 4°C storage conditions, ClO2 at a concentration of 15 ppm not only maintained the firmness and quality of tomatoes but also inhibited pitting during shelf-life with a concomitant increase of catalase activity. Moreover, treatment with 15 ppm ClO2 significantly reduced the calyx molding that is generally observed in fruits stored at 13°C and under shelf-life conditions. Hence, our results indicate that ClO2 treatment effectively maintained tomato quality and inhibited calyx molding by partially regulating ripening-related genes and antioxidant systems, thereby improving the storability of postharvest tomatoes.

The Als3 Cell Wall Adhesin Plays a Critical Role in Human Serum Amyloid A1-Induced Cell Death and Aggregation in Candida albicans.

Antimicrobial peptides and proteins play critical roles in the host defense against invading pathogens. We recently discovered that recombinantly expressed human and mouse serum amyloid A1 (rhSAA1 and rmSAA1, respectively) proteins have potent antifungal activities against the major human fungal pathogen Candida albicans At high concentrations, rhSAA1 disrupts C. albicans membrane integrity and induces rapid fungal cell death. In the present study, we find that rhSAA1 promotes cell aggregation and targets the C. albicans cell wall adhesin Als3. Inactivation of ALS3 in C. albicans leads to a striking decrease in cell aggregation and cell death upon rhSAA1 treatment, suggesting that Als3 plays a critical role in SAA1 sensing. We further demonstrate that deletion of the transcriptional regulators controlling the expression of ALS3, such as AHR1, BCR1, and EFG1, in C. albicans results in similar effects to that of the als3/als3 mutant upon rhSAA1 treatment. Global gene expression profiling indicates that rhSAA1 has a discernible impact on the expression of cell wall- and metabolism-related genes, suggesting that rhSAA1 treatment could lead to a nutrient starvation effect on C. albicans cells.

Expression of Cell Wall-Modifying Enzymes in Aspen for Improved Lignocellulose Processing.

Wood is an important source of biomass for materials and chemicals, and a target for genetic engineering of its properties for different applications or for research. Wood properties can be altered by using different enzymes acting on cell wall polymers postsynthetically in cell walls. This approach allows for a precise polymer structure modification thanks to the specificity of enzymes used. Such enzymes can originate from all kinds of organisms, or even be modified in a desired way for novel attributes. Here we present a general strategy for expressing a microbial enzyme in aspen and targeting it to cell wall, using an example of fungal glucuronoyl esterase. We describe methods of vector cloning, plant transformation, transgenic line selection and multiplication, testing for the presence of enzymatic activity in different cell compartments, and finally the method of plant transferring from sterile culture to the greenhouse conditions.

Immunoproteomics to identify Staphylococcus aureus antigens expressed in bovine milk during mastitis

Staphylococcus aureus is an opportunistic pathogen affecting both human and animal species. An effective vaccine to prevent S. aureus bovine disease and transmission would have positive effects on animal well-being, food production, and human health. The objective of this study was to identify multiple antigens that are immunoreactive during udder colonization and disease for exploration as vaccine antigens to prevent bovine mastitis. Staphylococcus aureus produces several cell wall-anchored and surface-associated virulence factors that play key roles in the pathogenesis of mastitis. Many of these proteins are conserved between different strains of S. aureus and represent promising vaccine candidates. We used an immunoproteomics approach to identify antigenic proteins from the surface of S. aureus. The expression of cell wall and surface proteins from S. aureus was induced under low iron conditions, followed by trypsin extraction and separation by 2-dimensional electrophoresis. The separated proteins were blotted with antibodies from mastitic bovine milk and identified by liquid chromatography-mass spectrometry. Thirty-eight unique proteins were identified, of which 8 were predicted to be surface exposed and involved in S. aureus virulence. Two surface proteins, iron-regulated surface determinant protein C (IsdC) and ESAT-6 secretion system extracellular protein (EsxA), were cloned, expressed, and purified from Escherichia coli for confirmation of immune reactivity by ELISA. A PCR of 37 bovine S. aureus isolates indicated that the presence of esxA and isdC is conserved, and amino acid alignments revealed that IsdC and EsxA sequences are highly conserved. The immunoproteomics technique used in this study generated reproducible results and identified surface exposed and reactive antigens for further characterization.

English

Melons are good models used for explaining the physiological and biochemical changes in postharvest ripening. In this study, transgenic melons expressing apple ACC oxidase gene when treated with ethylene (AS3) were evaluated. Cell wall expression (MPG1; MPG2), ethylene synthesis ACC oxidase (ACCO1), flavour synthesis AAT (alcohol acyltransferase) and physiochemical parameters such as pulp firmness, titratable acidity (TA), soluble solid content (SSC), volatile esters, ethylene productions, antioxidant assay capacity and ascorbic acid content were evaluated. In cell wall expression analysis, MPG1 increased when fruits of transgenic melons were exposed to ethylene; showing they are ethylene- dependent. MPG2 decreased gradually when fruits were subjected to ethylene application. Fruit firmness was modified in transgenic fruits when ethylene was applied. There was a great reduction similar to that of non- transgenic fruits. However, TA in transgenic fruits remained lower than in non- transgenic fruit. The ethylene applied in transgenic fruit made the titratable acid to increase during 48 h and after it, a reduction was observed. In relation to soluble solid contents, transgenic fruits treated with or without ethylene did not reduce gradually compared to the wild type melons in all the periods. Ethylene productions in transgenic fruits were reestablished when ethylene was applied, exhibiting the same behavior as transgenic fruits. Antioxidant assay levels were more active in transgenic fruits when ethylene was applied than in control fruits, and it was only in transgenic fruits without ethylene. Ascorbic acid was kept in transgenic fruits with or without the application of ethylene. Results obtained show that the application of ethylene in transgenic ACC oxidase melons is able to change the metabolism of the cell wall, flavors and antioxidant capacity levels in fruit during the ripening process. Keywords: Esters, antioxidant, solid soluble content, ascorbic acid African Journal of Biotechnology , Vol 13(31) 3252-3261

Local adaptation of an introduced transgenic insect fungal pathogen due to new beneficial mutations

Genetically modified Metarhizium spp represent a major new arsenal for combating insect pests and insect-borne diseases. However, for these tools to be used safely and effectively, we need a much better understanding of their evolutionary potential and invasion ecology. In order to model natural as well as anthropogenic dispersal scenarios, we investigated evolutionary processes in a green fluorescent protein tagged strain of Metarhizium robertsii following transfer from a semitropical to a temperate soil community. Adaptive changes occurred over four years despite recurrent genetic bottlenecks and lack of recombination with locally well adapted strains. By coupling microarray-based functional analysis with DNA hybridizations we determined that expression of cell wall and stress response genes evolved at an accelerated rate in multiple replicates, whereas virulence determinants, transposons, and chromosome structure were unaltered. The mutable genes were enriched for TATA boxes possibly because they are larger mutational targets. In further field trials, we showed that the new mutations increased the fitness of M. robertsii in the new range by enhancing saprophytic associations, and these benefits were maintained in subsequent years. Consistent with selection being habitat rather than host specific, populations of an avirulent mutant cycled with seasons similarly to the wild type, whereas a mutant unable to adhere to plant roots showed a linear decrease in population. Our results provide a mechanistic basis for understanding postrelease adaptations, show that agents can be selected that lack gene flow and virulence evolution, and describe a means of genetically containing transgenic strains by disrupting the Mad2 gene.

Connecting Mutations of the RNA Polymerase II C-Terminal Domain to Complex Phenotypic Changes Using Combined Gene Expression and Network Analyses

The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA synthesis and processing, through coordination of an astounding array of protein-protein interactions. Not surprisingly, CTD mutations can have complex, pleiotropic impacts on phenotype. For example, insertions of five alanine residues between CTD diheptads in yeast, which alter the CTD's overall tandem structure and physically separate core functional units, dramatically reduce growth rate and result in abnormally large cells that accumulate increased DNA content over time. Patterns by which specific CTD-protein interactions are disrupted by changes in CTD structure, as well as how downstream metabolic pathways are impacted, are difficult to target for direct experimental analyses. In an effort to connect an altered CTD to complex but quantifiable phenotypic changes, we applied network analyses of genes that are differentially expressed in our five alanine CTD mutant, combined with established genetic interactions from the Saccharomyces cerevisiae Genome Database (SGD). We were able to identify candidate genetic pathways, and several key genes, that could explain how this change in CTD structure leads to the specific phenotypic changes observed. These hypothetical networks identify links between CTD-associated proteins and mitotic function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. Such results can help to direct future genetic and biochemical investigations that tie together the complex impacts of the CTD on global cellular metabolism.

Orchestration of transpiration, growth and carbohydrate dynamics in rice during a dry-down cycle.

The regulation of carbohydrate metabolism and source-sink relationships among organs play a key role in plant adaptation to drought. This study aimed at characterising the dynamics of transpiration, development, growth and carbon metabolism, as well as the expression of invertase genes, in response to drought during a dry-down cycle. Three 1-month experiments were conducted in controlled environment using the rice genotype IR64 (Oryza sativa L., indica). Plant leaf relative transpiration and expansion rates decreased linearly when fraction of transpirable soil water (FTSW) dropped below 0.66 and 0.58, respectively. Hexose and starch concentration responses to FTSW in a given organ were generally linear and opposite: in source leaves, hexose concentration increased and starch decreased, and vice versa in sink leaves and roots. Sucrose remained constant in source leaves and increased slightly in sink leaves. Starch reserves built up during stress in sink organs were rapidly mobilised upon rewatering, indicating its involvement in a mechanism to ensure recovery. Expression of cell-wall and vacuolar invertase genes under stress increased in sink leaves, interpreted as a mechanism to maintain sink activity (cell wall) and osmotic adjustment (vacuolar). It is concluded that carbohydrate metabolism in sink organs under drought is highly regulated, and important for stress adaptation.

Characterization of the sar locus and its interaction with agr in Staphylococcus aureus.

The expression of cell wall and extracellular proteins in Staphylococcus aureus is controlled by global regulatory systems, including sar and agr. We have previously shown that a transposon insertion into the 372-bp sarA gene within the sar locus resulted in decreased expression of several extracellular and cell wall proteins (A. L. Cheung and S. J. Projan, J. Bacteriol. 176:4168-4172, 1994). In this study, Northern (RNA blot) analysis with a 732-bp sarA probe indicated that two major transcripts (0.56 and 1.2 kb) were absent in the sar mutant compared with the parental strain RN6390. Additional transcriptional studies revealed that the sarA gene is encoded within the 0.56-kg transcript. Notably, a plasmid carrying the sarA gene together with a 1.2-kb upstream fragment (1.7 kb total) was able to reestablish the 1.2-kb transcript in the mutant. Although reconstitution of the parental phenotype by the sarA gene was incomplete, the introduction of a plasmid carrying the 1.7-kb fragment to the mutant restored the parental phenotype. Transcription of RNAII and RNAIII, which encode the structural and regulatory genes of agr, respectively, was diminished in the mutant but restored to wild-type levels by complementation with the 1.7-kb fragment. In gel shift assays, cell extracts of this clone were able to retard the mobility of a labeled RNAII promoter probe but not an RNAIII promoter element. These data suggest that sarA and the adjacent upstream DNA are essential to the expression of a DNA-binding protein(s) with specificity for the RNAII promoter, thereby controlling agr-related transcription.