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1.
Microbiol Res ; 230: 126348, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31639624

RESUMO

The transcription factor PacC/Rim101 participates in environmental pH adaptation, development and secondary metabolism in many fungi, but whether PacC/Rim101 contributes to fungal adaptation to environmental stress remains unclear. In our previous study, a homologous gene of PacC/Rim101 was identified, and PacC-silenced strains of the agaricomycete Ganoderma lucidum were constructed. In this study, we further investigated the functions of PacC in G. lucidum and found that PacC-silenced strains were hypersensitive to environmental stresses, such as osmotic stress, oxidative stress and cell wall stress, compared with wild-type (WT) and empty-vector control (CK) strains. In addition, transmission electron microscopy images of the cell wall structure showed that the cell walls of the PacC-silenced strains were thinner (by approximately 25-30%) than those of the WT and CK strains. Further analysis of cell wall composition showed that the ß-1,3-glucan content in the PacC-silenced strains was only approximately 78-80% of that in the WT strain, and the changes in ß-1,3-glucan content were consistent with downregulation of glucan synthase gene expression. The ability of PacC to bind to the promoters of glucan synthase-encoding genes confirms that PacC transcriptionally regulates these genes.


Assuntos
Parede Celular/química , Proteínas Fúngicas/metabolismo , Reishi/metabolismo , Fatores de Transcrição/metabolismo , Adaptação Fisiológica , Parede Celular/genética , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Estresse Oxidativo , Reishi/genética , Fatores de Transcrição/genética , beta-Glucanas/química , beta-Glucanas/metabolismo
2.
BMC Genomics ; 20(1): 785, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664907

RESUMO

BACKGROUND: The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems. RESULTS: High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize (Zea mays, cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at ≥95 reads per 20 M, and 693 of them at ≥500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements. CONCLUSIONS: Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.


Assuntos
Parede Celular/genética , Caules de Planta/genética , Transcriptoma , Zea mays/genética , Arabidopsis/genética , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Celulose/biossíntese , Lignina/biossíntese , Família Multigênica , Melhoramento Vegetal , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Regiões Promotoras Genéticas , Xilanos/biossíntese , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Zea mays/ultraestrutura
3.
PLoS Biol ; 17(10): e3000512, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658248

RESUMO

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.


Assuntos
Arrestinas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Nucleosídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tiamina/metabolismo , Arrestinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Endocitose/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Proteínas de Transporte de Nucleosídeos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Secundária de Proteína , Proteômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Tiamina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação
4.
Nat Commun ; 10(1): 4797, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641111

RESUMO

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Silenced cells exhibit a reduced or peeled-off S-layer lattice, cell shape alterations and decreased surface glycosylation. These cells barely propagate but increase in diameter and DNA content, indicating impaired cell division; their phenotypes can be rescued through genetic complementation. Furthermore, S-layer depleted cells are less susceptible to infection with the virus SSV1. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is important for cell division and virus susceptibility.


Assuntos
Proteínas Arqueais/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sulfolobus solfataricus/citologia , Sulfolobus solfataricus/virologia , Proteínas Arqueais/genética , Parede Celular/genética , Parede Celular/metabolismo , Cromossomos de Archaea , Fuselloviridae , Técnicas de Silenciamento de Genes , Inativação Gênica , Teste de Complementação Genética , Glicosilação , Interações Hospedeiro-Patógeno/genética , Sulfolobus solfataricus/genética
5.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615142

RESUMO

Manganese (Mn) is an essential element for plant growth due to its participation in a series of physiological and metabolic processes. Mn is also considered a heavy metal that causes phytotoxicity when present in excess, disrupting photosynthesis and enzyme activity in plants. Thus, Mn toxicity is a major constraint limiting plant growth and production, especially in acid soils. To cope with Mn toxicity, plants have evolved a wide range of adaptive strategies to improve their growth under this stress. Mn tolerance mechanisms include activation of the antioxidant system, regulation of Mn uptake and homeostasis, and compartmentalization of Mn into subcellular compartments (e.g., vacuoles, endoplasmic reticulum, Golgi apparatus, and cell walls). In this regard, numerous genes are involved in specific pathways controlling Mn detoxification. Here, we summarize the recent advances in the mechanisms of Mn toxicity tolerance in plants and highlight the roles of genes responsible for Mn uptake, translocation, and distribution, contributing to Mn detoxification. We hope this review will provide a comprehensive understanding of the adaptive strategies of plants to Mn toxicity through gene regulation, which will aid in breeding crop varieties with Mn tolerance via genetic improvement approaches, enhancing the yield and quality of crops.


Assuntos
Transporte Biológico/efeitos dos fármacos , Manganês/toxicidade , Metais Pesados/toxicidade , Plantas/genética , Antioxidantes/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Homeostase/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Plantas/efeitos dos fármacos
6.
PLoS Genet ; 15(10): e1008263, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626625

RESUMO

In Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in yidC1 (spoIIIJ), which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 YidC1 (SpoIIIJ) contains a single amino acid substitution in a functionally important hydrophilic groove (Q140K), and this allele suppresses the lethality of high σM. Analysis of a library of YidC1 variants reveals that increased charge (+2 or +3) in the hydrophilic groove can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1Q140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of the SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.


Assuntos
Bacillus subtilis/genética , Integrases/genética , Proteínas de Membrana Transportadoras/genética , Fator sigma/genética , Bacillus subtilis/crescimento & desenvolvimento , Membrana Celular/genética , Parede Celular/genética , Dano ao DNA/genética , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Óperon/genética , Regiões Promotoras Genéticas , Regulon/genética
7.
J Microbiol Biotechnol ; 29(11): 1806-1816, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31546294

RESUMO

Candida albicans is an opportunistic fungus possessing multiple virulence factors controlling pathogenicity. Cell wall proteins are the most important among these factors, being the first elements contacting the host. Ddr48 is a cell wall protein consisting of 212 amino acids. A DDR48 haploinsufficient mutant strain was previously found necessary for proper oxidative stress response and drug resistance. In this study, we aimed to further elucidate the role of Ddr48 by performing additional phenotypic characterization assays. A combinatory proteomic and bioinformatics approach was also undertaken to determine differentially expressed cell wall proteins. Results showed that the mutant strain exhibited a 10% decrease in adhesion mirrored by a 20% decrease in biofilm formation, and slight sensitivity to menadione, diamide, and SDS. Both strains showed similar hyphae formation, virulence, temperature tolerance, and calcofluor white and Congo red sensitivities. Furthermore, a total of 8 and 10 proteins were identified exclusively in the wild-type strain grown under filamentous and nonfilamentous conditions respectively. Results included proteins responsible for superoxide stress resistance (Sod4 and Sod6), adhesion (Als3, Hyr4, Pmt1, and Utr2), biofilm formation (Hsp90, Ece1, Rim9, Ipp1, and Pra1) and cell wall integrity (Utr2 and Pga4). The lack of detection of these proteins in the mutant strain correlates with the observed phenotypes.


Assuntos
Candida albicans/fisiologia , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Estresse Oxidativo/genética , Fatores de Virulência/genética , Aderência Bacteriana/genética , Biofilmes/crescimento & desenvolvimento , Candida albicans/genética , Candida albicans/metabolismo , Parede Celular/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/metabolismo , Mutação , Fenótipo , Proteômica , Fatores de Virulência/metabolismo
8.
Genes (Basel) ; 10(9)2019 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500311

RESUMO

Wood, the most abundant biomass on Earth, is composed of secondary xylem differentiated from vascular cambium. However, the underlying molecular mechanisms of wood formation remain largely unclear. To gain insight into wood formation, we performed a series of wood-forming tissue-specific transcriptome analyses from a hybrid poplar (Populus alba × P. glandulosa, clone BH) using RNA-seq. Together with shoot apex and leaf tissue, cambium and xylem tissues were isolated from vertical stem segments representing a gradient of secondary growth developmental stages (i.e., immature, intermediate, and mature stem). In a comparative transcriptome analysis of the 'developing xylem' and 'leaf' tissue, we could identify critical players catalyzing each biosynthetic step of secondary wall components (e.g., cellulose, xylan, and lignin). Several candidate genes involved in the initiation of vascular cambium formation were found via a co-expression network analysis using abundantly expressed genes in the 'intermediate stem-derived cambium' tissue. We found that transgenic Arabidopsis plants overexpressing the PtrHAM4-1, a GRAS family transcription factor, resulted in a significant increase of vascular cambium development. This phenotype was successfully reproduced in the transgenic poplars overexpressing the PtrHAM4-1. Taken together, our results may serve as a springboard for further research to unravel the molecular mechanism of wood formation, one of the most important biological processes on this planet.


Assuntos
Câmbio/genética , Parede Celular/genética , Populus/genética , Transcriptoma , Câmbio/crescimento & desenvolvimento , Parede Celular/metabolismo , Lignina/biossíntese , Lignina/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Populus/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xilanos/biossíntese , Xilanos/genética , Xilema/genética , Xilema/crescimento & desenvolvimento
9.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31400094

RESUMO

Mycobacteria, including the infamous pathogen Mycobacterium tuberculosis, are high-GC Gram-positive bacteria with a distinctive cell envelope. Although there is a typical inner membrane, the mycobacterial cell envelope is unusual in having its peptidoglycan layer connected to a polymer of arabinogalactan, which in turn is covalently attached to long-chain mycolic acids that help form a highly impermeable mycobacterial outer membrane. This complex double-membrane, or diderm, cell envelope imparts mycobacteria with unique requirements for protein export into and across the cell envelope for secretion into the extracellular environment. In this article, we review the four protein export pathways known to exist in mycobacteria: two conserved systems that exist in all types of bacteria (the Sec and Tat pathways) and two specialized systems that exist in mycobacteria, corynebacteria, and a subset of low-GC Gram-positive bacteria (the SecA2 and type VII secretion pathways). We describe the progress made over the past 15 years in understanding each of these mycobacterial export pathways, and we highlight the need for research to understand the specific steps of protein export across the mycobacterial outer membrane.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Mycobacterium tuberculosis/metabolismo , Animais , Proteínas de Bactérias/genética , Membrana Celular/genética , Parede Celular/genética , Humanos , Mycobacterium tuberculosis/genética , Transporte Proteico , Tuberculose/microbiologia
10.
PLoS Genet ; 15(8): e1008296, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31437162

RESUMO

The peptidoglycan (PG) sacculus is composed of long glycan strands cross-linked together by short peptides forming a covalently closed meshwork that protects the bacterial cell from osmotic lysis and specifies its shape. PG hydrolases play essential roles in remodeling this three-dimensional network during growth and division but how these autolytic enzymes are regulated remains poorly understood. The FtsEX ABC transporter-like complex has emerged as a broadly conserved regulatory module in controlling cell wall hydrolases in diverse bacterial species. In most characterized examples, this complex regulates distinct PG hydrolases involved in cell division and is intimately associated with the cytokinetic machinery called the divisome. However, in the gram-positive bacterium Bacillus subtilis the FtsEX complex is required for cell wall elongation where it regulates the PG hydrolase CwlO that acts along the lateral cell wall. To investigate whether additional factors are required for FtsEX function outside the divisome, we performed a synthetic lethal screen taking advantage of the conditional essentiality of CwlO. This screen identified two uncharacterized factors (SweD and SweC) that are required for CwlO activity. We demonstrate that these proteins reside in a membrane complex with FtsX and that amino acid substitutions in residues adjacent to the ATPase domain of FtsE partially bypass the requirement for them. Collectively our data indicate that SweD and SweC function as essential co-factors of FtsEX in controlling CwlO during cell wall elongation. We propose that factors analogous to SweDC function to support FtsEX activity outside the divisome in other bacteria.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Divisão Celular/genética , Parede Celular/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Elementos de DNA Transponíveis/genética , Mutação , Peptidoglicano/metabolismo
11.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31373270

RESUMO

Surface proteins are critical for the survival of gram-positive bacteria both in the environment and to establish an infection. Depending on the organism, their surface proteins are evolutionarily tailored to interact with specific ligands on their target surface, be it inanimate or animate. Most surface molecules on these organisms are covalently anchored to the peptidoglycan through an LPxTG motif found at the C-terminus. These surface molecules are generally modular with multiple binding or enzymatic domains designed for a specific survival function. For example, some molecules will bind serum proteins like fibronectin or fibrinogen in one domain and have a separate function in another domain. In addition, enzymes such as those responsible for the production of ATP may be generally found on some bacterial surfaces, but when or how they are used in the life of these bacteria is currently unknown. While surface proteins are required for pathogenicity but not viability, targeting the expression of these molecules on the bacterial surface would prevent infection but not death of the organism. Given that the number of different surface proteins could be in the range of two to three dozen, each with two or three separate functional domains (with hundreds to thousands of each protein on a given organism), exemplifies the complexity that exists on the bacterial surface. Because of their number, we could not adequately describe the characteristics of all surface proteins in this chapter. However, since the streptococcal M protein was one of the first gram-positive surface protein to be completely sequenced, and perhaps one of the best studied, we will use M protein as a model for surface proteins in general, pointing out differences with other surface molecules when necessary.


Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Positivas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Parede Celular/química , Parede Celular/genética , Parede Celular/metabolismo , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/genética , Domínios Proteicos
12.
Genetics ; 213(1): 213-227, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31266771

RESUMO

Limited antifungal diversity and availability are growing problems for the treatment of fungal infections in the face of increasing drug resistance. The echinocandins, one of the newest classes of antifungal drugs, inhibit production of a crucial cell wall component. However, these compounds do not effectively inhibit the growth of the opportunistic fungal pathogen Cryptococcus neoformans, despite potent inhibition of the target enzyme in vitro Therefore, we performed a forward genetic screen to identify cellular processes that mediate the relative tolerance of this organism to the echinocandin drug caspofungin. Through these studies, we identified 14 genetic mutants that enhance caspofungin antifungal activity. Rather than directly affecting caspofungin antifungal activity, these mutations seem to prevent the activation of various stress-induced compensatory cellular processes. For example, the pfa4Δ mutant has defects in the palmitoylation and localization of many of its target proteins, including the Ras1 GTPase and the Chs3 chitin synthase, which are both required for caspofungin tolerance. Similarly, we have confirmed the link between caspofungin treatment and calcineurin signaling in this organism, but we suggest a deeper mechanism in which caspofungin tolerance is mediated by multiple pathways downstream of calcineurin function. In summary, we describe here several pathways in C. neoformans that contribute to the complex caspofungin tolerance phenotype in this organism.


Assuntos
Antifúngicos/farmacologia , Caspofungina/farmacologia , Parede Celular/genética , Cryptococcus neoformans/genética , Farmacorresistência Fúngica/genética , Genes Fúngicos , Calcineurina/genética , Calcineurina/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Estresse Fisiológico , Proteínas ras/genética , Proteínas ras/metabolismo
13.
World J Microbiol Biotechnol ; 35(7): 105, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267317

RESUMO

Pseudocercospora fijiensis causes black Sigatoka disease, the most important threat to banana. The cell wall is crucial for fungal biological processes, including pathogenesis. Here, we performed cell wall proteomics analyses of two P. fijiensis strains, the highly virulent Oz2b, and the less virulent C1233 strains. Strains were starved from nitrogen to mimic the host environment. Interestingly, in vitro cultures of the C1233 strain grew faster than Oz2b in PDB medium, suggesting that C1233 survives outside the host better than the highly virulent Oz2b strain. Both strains were submitted to nitrogen starvation and the cell wall proteins were isolated and subjected to nano-HPLC-MS/MS. A total of 2686 proteins were obtained from which only 240 had a known function and thus, bioinformatics analyses were performed on this group. We found that 90 cell wall proteins were shared by both strains, 21 were unique for Oz2b and 39 for C1233. Shared proteins comprised 24 pathogenicity factors, including Avr4 and Ecp6, two effectors from P. fijiensis, while the unique proteins comprised 16 virulence factors in C1233 and 11 in Oz2b. The P. fijiensis cell wall proteome comprised canonical proteins, but thirty percent were atypical, a feature which in other phytopathogens has been interpreted as contamination. However, a comparison with the identities of atypical proteins in other reports suggests that the P. fijiensis proteins we detected were not contaminants. This is the first proteomics analysis of the P. fijiensis cell wall and our results expands the understanding of the fundamental biology of fungal phytopathogens and will help to decipher the molecular mechanisms of pathogenesis and virulence in P. fijiensis.


Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Proteoma , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Ascomicetos/isolamento & purificação , Ascomicetos/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Genoma Fúngico , Musa/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Espectrometria de Massas em Tandem , Virulência
14.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31267890

RESUMO

The highly cross-linked peptidoglycan represents the rigid layer of the bacterial envelope and protects bacteria from osmotic lysis. In Gram-positive bacteria, peptidoglycan also functions as a scaffold for the immobilization of capsular polysaccharide, wall teichoic acid (WTA), and surface proteins. This chapter captures recent development on the assembly of the envelope of Staphylococcus aureus including mechanisms accounting for immobilization of molecules to peptidoglycan as well as hydrolysis of peptidoglycan for the specific release of bound molecules, facilitation of protein secretion across the envelope and cell division. Peptidoglycan, WTA and capsular polysaccharide are directly synthesized onto undecaprenol. Surface proteins are anchored by Sortase A, a membrane-embedded transpeptidase that scans secreted polypeptides for the C-terminal LPXTG motif of sorting signals. The resulting acyl enzyme intermediate is resolved by lipid II, the undecaprenol-bound peptidoglycan precursor. While these pathways share membrane diffusible undecaprenol, assembly of these molecules occurs either at the cross-walls or the cell poles. In S. aureus, the cross-wall represents the site of de novo peptidoglycan synthesis which is eventually split to complete the cell cycle yielding newly divided daughter cells. Peptidoglycan synthesized at the cross-wall is initially devoid of WTA. Conversely, lipoteichoic acid (LTA) synthesis which does not require bactoprenol is seemingly restricted to septal membranes. Similarly, S. aureus distinguishes two types of surface protein precursors. Polypeptides with canonical signal peptides are deposited at the cell poles, whereas precursors with conserved YSIRK-GXXS motif signal peptides traffic to the cross-wall. A model for protein trafficking in the envelope and uneven distribution of teichoic acids is discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/genética , Membrana Celular/genética , Parede Celular/genética , Parede Celular/metabolismo , Humanos , Proteínas de Membrana/genética , Peptidoglicano/metabolismo , Transporte Proteico , Staphylococcus aureus/genética
15.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31267927

RESUMO

The mycobacterial cell envelope consists of a typical plasma membrane of lipid and protein surrounded by a complex cell wall composed of carbohydrate and lipid. In pathogenic species, such as Mycobacterium tuberculosis, an outermost "capsule" layer surrounds the cell wall. This wall embraces a fundamental, covalently linked "cell-wall skeleton" composed of peptidoglycan, solidly attached to arabinogalactan, whose penta-saccharide termini are esterified by very-long-chain fatty acids (mycolic acids). These fatty acids form the inner leaflet of an outer membrane, called the mycomembrane, whose outer leaflet consists of a great variety of non-covalently linked lipids and glycolipids. The thickness of the mycomembrane, which is similar to that of the plasma membrane, is surprising in view of the length of mycoloyl residues, suggesting dedicated conformations of these fatty acids. Finally, a periplasmic space also exists in mycobacteria, between the plasma membrane and the peptidoglycan. This article provides a comprehensive overview of this biologically important and structurally unique mycobacterial cell compartment.


Assuntos
Membrana Celular/química , Parede Celular/química , Mycobacterium tuberculosis/metabolismo , Animais , Membrana Celular/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Galactanos/química , Galactanos/metabolismo , Glicolipídeos/química , Glicolipídeos/metabolismo , Humanos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Peptidoglicano/química , Peptidoglicano/metabolismo , Tuberculose/microbiologia
16.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31322105

RESUMO

Dating back to the 1960s, initial studies on the staphylococcal cell wall were driven by the need to clarify the mode of action of the first antibiotics and the resistance mechanisms developed by the bacteria. During the following decades, the elucidation of the biosynthetic path and primary composition of staphylococcal cell walls was propelled by advances in microbial cell biology, specifically, the introduction of high-resolution analytical techniques and molecular genetic approaches. The field of staphylococcal cell wall gradually gained its own significance as the complexity of its chemical structure and involvement in numerous cellular processes became evident, namely its versatile role in host interactions, coordination of cell division and environmental stress signaling.This chapter includes an updated description of the anatomy of staphylococcal cell walls, paying particular attention to information from the last decade, under four headings: high-resolution analysis of the Staphylococcus aureus peptidoglycan; variations in peptidoglycan composition; genetic determinants and enzymes in cell wall synthesis; and complex functions of cell walls. The latest contributions to a more precise picture of the staphylococcal cell envelope were possible due to recently developed state-of-the-art microscopy and spectroscopy techniques and to a wide combination of -omics approaches, that are allowing to obtain a more integrative view of this highly dynamic structure.


Assuntos
Parede Celular/química , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/citologia , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Humanos , Peptidoglicano/química , Peptidoglicano/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
17.
Int J Nanomedicine ; 14: 4801-4816, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308659

RESUMO

Background: Silver nanoparticles (AgNPs) inhibit the proliferation of various fungi; however, their mechanisms of action remain poorly understood. To better understand the inhibitory mechanisms, we focused on the early events elicited by 5 nm AgNPs in pathogenic Candida albicans and non-pathogenic Saccharomyces cerevisiae. Methods: The effect of 5 nm and 100 nm AgNPs on fungus cell proliferation was analyzed by growth kinetics monitoring and spot assay. We examined cell cycle progression, reactive oxygen species (ROS) production, and cell death using flow cytometry. Glucose uptake was assessed using tritium-labeled 2-deoxyglucose. Results: The growth of both C. albicans and S. cerevisiae was suppressed by treatment with 5 nm AgNPs but not with 100 nm AgNPs. In addition, 5 nm AgNPs induced cell cycle arrest and a reduction in glucose uptake in both fungi after 30 minutes of culture in a dose-dependent manner (P<0.05). However, in C. albicans only, an increase in ROS production was detected after exposure to 5 nm AgNPs. Concordantly, an ROS scavenger blocked the effect of 5 nm AgNPs on the cell cycle and glucose uptake in C. albicans only. Furthermore, the growth-inhibition effect of 5 nm AgNPs was not greater in S. cerevisiae mutant strains deficient in oxidative stress response genes than it was in wild type. Finally, 5 nm AgNPs together with a glycolysis inhibitor, 3-bromopyruvate, synergistically enhanced cell death in C. albicans (P<0.05) but not in S. cerevisiae. Conclusion: AgNPs exhibit antifungal activity in a manner that may or may not be ROS dependent, according to the fungal species. The combination of AgNPs with 3-bromopyruvate may be more useful against infection with C. albicans.


Assuntos
Candida albicans/citologia , Ciclo Celular/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Piruvatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/citologia , Prata/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Depuradores de Radicais Livres/farmacologia , Fase G1/efeitos dos fármacos , Genes Fúngicos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
18.
Plant Physiol Biochem ; 142: 106-116, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31279135

RESUMO

This study presents evidence that strigolactones (SLs) promote defense against devastating rice blast fungal pathogen Magnaporthe oryzae. Impairment in either SL-biosynthetic dwarf17 (d17) or -signaling (d14) led to increased susceptibility towards M. oryzae. Comparative transcriptome profiling of the SL-signaling d14 mutant and WT plants revealed that a large number of defense-associated genes including hydrogen peroxide (H2O2)-, ethylene- and cell wall-synthesis-related genes were remarkably suppressed in d14 with respect to that of WT plants, during M. oryzae infection. In addition, various KEGG metabolic pathways related to sugar metabolism were significantly suppressed in the d14 plants compared to WT, during M. oryzae infection. Accordingly, WT plants accumulated increased levels of H2O2 and soluble sugar content compared to that of d17 and d14 in response to M. oryzae infection. Altogether, these results propose that SLs positively regulated rice defense against M. oryzae through involvement in the induction of various defense associated genes/pathways.


Assuntos
Lactonas/metabolismo , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/microbiologia , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Lactonas/farmacologia , Mutação , Oryza/efeitos dos fármacos , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Transdução de Sinais/genética , Açúcares/metabolismo
19.
Int J Mol Sci ; 20(14)2019 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-31337083

RESUMO

Histone acetylation and deacetylation play essential roles in eukaryotic gene regulation. HD2 (HD-tuins) proteins were previously identified as plant-specific histone deacetylases. In this study, we investigated the function of the HDT1 gene in the formation of stem vascular tissue in Arabidopsis thaliana. The height and thickness of the inflorescence stems in the hdt1 mutant was lower than that of wild-type plants. Paraffin sections showed that the cell number increased compared to the wild type, while transmission electron microscopy showed that the size of individual tracheary elements and fiber cells significantly decreased in the hdt1 mutant. In addition, the cell wall thickness of tracheary elements and fiber cells increased. We also found that the lignin content in the stem of the hdt1 mutants increased compared to that of the wild type. Transcriptomic data revealed that the expression levels of many biosynthetic genes related to secondary wall components, including cellulose, lignin biosynthesis, and hormone-related genes, were altered, which may lead to the altered phenotype in vascular tissue of the hdt1 mutant. These results suggested that HDT1 is involved in development of the vascular tissue of the stem by affecting cell proliferation and differentiation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Histona Desacetilases/genética , Desenvolvimento Vegetal/genética , Caules de Planta/genética , Feixe Vascular de Plantas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/metabolismo , Lignina/metabolismo , Mutação , Fenótipo , Caules de Planta/metabolismo , Feixe Vascular de Plantas/metabolismo , Xilema/citologia , Xilema/genética , Xilema/metabolismo
20.
J Sci Food Agric ; 99(15): 6689-6695, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31350766

RESUMO

BACKGROUND: Tomato fruit is susceptible to Alternaria spp. spoilage. Correct postharvest management is necessary to prevent mold growth and mycotoxin accumulation, temperature being one of the main factors associated with these problems. The effect of different postharvest temperatures (5, 12, 25, and 35 °C) on growth, mycotoxin production, and stress-related gene expression by two Alternaria spp. was assessed. RESULTS: Growth rates decreased rapidly when temperature was higher than the optimum (25 °C), while a gradual reduction was detected at lower temperatures. Tenuazonic acid (TeA) was strongly synthesized at all the temperatures that were evaluated, with a maximum between 12 and 25 °C. Alternariol monomethyl ether (AME) was produced only at the two lowest temperatures, with a peak at 12 °C. Regarding the expression of the stress-related RHO1 gene, during active fungal growth both Alternaria spp. showed more copies of the gene as temperature increased. At the stationary phase, RHO1 gene expression was significantly higher at 12 °C, coinciding with the highest accumulation of AME. CONCLUSION: Changes in temperature related to different postharvest stages of tomato fruits markedly affect toxigenic Alternaria spp. The highest levels of both mycotoxins were recorded at 12 °C, a common storage temperature for tomato fruit. An association between alternariol biosynthesis and the cell wall integrity pathway was also noticed in relation to temperature, suggesting that temperature may act as a stressor stimulating the RHO1 gene expression, which in turn triggers this mycotoxin synthesis. These results will be useful in developing new strategies to control Alternaria spoilage efficiently in tomato fruit and by-products. © 2019 Society of Chemical Industry.


Assuntos
Alternaria/crescimento & desenvolvimento , Alternaria/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Lycopersicon esculentum/microbiologia , Micotoxinas/biossíntese , Alternaria/genética , Parede Celular/genética , Frutas/química , Frutas/microbiologia , Proteínas Fúngicas/metabolismo , Lycopersicon esculentum/química , Temperatura Ambiente
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