Structural and functional characterization of proteins related to the pathogenicity of Xylella fastidiosa

Orientadores: Anete Pereira de Souza, Ricardo AparícioTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Xylella fastidiosa é uma bactéria responsável por inúmeras doenças de plantas em culturas economicamente importantes ao redor do mundo, incluindo a clorose variegada dos citros. Após a infecção de seu hospedeiro, as células de X. fastidiosa é apta a formarem uma estrutura de biofilme que bloqueia os vasos xilemáticos, levando a uma condição de estresse hídrico na planta hospedeira e desencadeando o desenvolvimento da doença. Tendo como estímulo a relevância econômica da citricultura para o Brasil e, visando reduzir os prejuízos provocados pelos problemas fitossanitários que acometem esta cultura, foi realizado um consórcio de pesquisa com o intuito de se conhecer completamente o genoma da linhagem 9a5c de X. fastidiosa. Inúmeras proteínas associadas com patogenicidade, adaptação e sobrevivência bacteriana foram identificadas, incluindo XfDsbC (proteína disulfeto isomerase), Xf5'-Nt (5'-nucelotidase), XfTolB (proteína de translocação B) e XfPal (lipoproteína associada ao peptidoglicano) que foram caracterizadas neste estudo. Empregando ferramentas de caracterização de proteínas, aspectos funcionais e estruturais destas quatro proteínas alvos foram avaliados. Dentre os resultados destaca-se a imunodetecção de XfDsbC, Xf5'-Nt, XfTolB e XfPal durante as diferentes fases de formação e desenvolvimento do biofilme de X. fastidiosa, que é tido como o principal mecanismo de patogenicidade deste fitopatógeno, confirmando a predição inicial de tais proteínas como associadas à patogenicidade bacteriana. Adicionalmente, resultados funcionais e estruturais revelaram detalhes finos do papel biológico desempenhado por cada uma das proteínas estudadas. Juntos, os resultados apresentados neste trabalho contribuem para o melhor entendimento de patogenicidade bacteriana, especialmente com respeito ao fitopatógeno X. fastidiosaAbstract: Xylella fastidiosa is a plant pathogen bacterium responsible for numerous economically important crops diseases around the world, including the citrus variegated chlorosis. Following the host infection, the X. fastidiosa cells are able to form a biofilm structure which block the xylem vessels, leading to a hydric stress condition in the host plant and triggers the disease development. Given the economic relevance of citriculture for Brazil and in order to reduce the damage caused by phytosanitary problems that affect the citrus production, a research consortium was established with the aim to elucidate the complete genome sequence of the X. fastidiosa 9a5c strain. Numerous proteins associated with bacterial pathogenicity, adaptation and survival have been identified, including XfDsbC (protein disulfide isomerase), Xf5'-Nt (5'-nucleotidase), XfTolB (protein translocation B) and XfPal (peptidoglycan-associated lipoprotein) which were characterized in this study. Using tools for protein characterization, structural and functional aspects of these four protein targets were evaluated. Among the results, we highlight the immunodetection of XfDsbC, Xf5'-Nt, XfTolB and XfPal during the different stages of X. fastidiosa biofilm formation and development which is considered the primary mechanism of pathogenicity of this pathogen. These findings, confirming the initial prediction that relates such proteins as associated with bacterial pathogenicity. Additionally, structural and functional results revealed accurate details of the biological role played by each protein studied. Taken together, the findings presented in this study contribute to a better understanding of bacterial pathogenesis, especially with regard to the plant pathogen X. fastidiosaDoutoradoGenetica de MicroorganismosDoutor em Genetica e Biologia Molecula

Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry

Abstract Background Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. Results In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Conclusions Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins

Business Models with Additive Manufacturing : opportunities and Challenges from the Perspective of Economics and Management

Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes

GO term distribution among the annotated BAC sequences.

<p><b>(A)</b> According to biological processes; (<b>B)</b> According to molecular functions; and <b>(C)</b> According to cellular components.</p

Species distribution.

<p>Based on the number of BLAST hits for predicted genes, <i>T</i>. <i>virens</i> is the species with the most similarity to <i>T</i>. <i>harzianum</i>.</p

Insert size distribution in the <i>T</i>. <b><i>harzianum</i> IOC-3844 BAC library</b>.

<p>Sixty randomly selected BAC clones were digested by <i>Not</i>I. The insert size ranged from 35 to 180 kbp, with an average size of 90 kbp.</p

Hierarchical clustering of CAZy genes.

<p>CAZy sequences were mapped against transcriptome reads and divided into 5 transcript groups according to expression levels. Group IV contains the most highly expressed sequences, and group I contains the sequences with the lowest expression.</p

Schematic model of CAZy and co-regulated genes.

<p>CAZy genes are indicated with color-filled arrows. Co-regulated non-CAZy genes are indicated by white arrows with contours of the same color as those of CAZy genes with similar profiles. Similar colors indicate a similar expression profile. (A) The region of the Egl1 BAC containing Egl1 and a gene encoding a GH71 family protein, which exhibit similar expression profiles. (B) The region of the Egl2 BAC containing a GH3 family protein, an Egl2 gene, and a GH79 protein. (C) The region of the Egl3 BAC containing 5 CAZy genes: CE16, GT90, Egl3, CH37, and GT17. (D) The region of the Bgl2 BAC that contains only one CAZy, the Bgl2 gene. (E) The Cbh1 BAC, demonstrating that Cbh1 and swollenin are co-induced. Two other CAZy genes (GH92 and GCase) are also present. (F) The region of the Xyn2 BAC containing a GH27 protein and <i>Xy2</i>.</p