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1.
PLoS One ; 15(12): e0243867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33338036

RESUMO

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ß subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.


Assuntos
Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Proteômica , Xanthomonas/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono/metabolismo , Genes Bacterianos , Anotação de Sequência Molecular , Fosfoglucomutase/metabolismo , Reprodutibilidade dos Testes , Xanthomonas/enzimologia , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
4.
PLoS One ; 15(3): e0231024, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32203563

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0162886.].

5.
PLoS One ; 14(1): e0209988, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30615696

RESUMO

Citrus canker is a plant disease caused by the bacteria Xanthomonas citri subsp. citri that affects all domestic varieties of citrus. Some annotated genes from the X. citri subsp. citri genome are assigned to an interesting class named "pathogenicity, virulence and adaptation". Amongst these is sodM, which encodes for the gene product XcSOD, one of four superoxide dismutase homologs predicted from the genome. SODs are widespread enzymes that play roles in the oxidative stress response, catalyzing the degradation of the deleterious superoxide radical. In Xanthomonas, SOD has been associated with pathogenesis as a counter measure against the plant defense response. In this work we initially present the 1.8 Å crystal structure of XcSOD, a manganese containing superoxide dismutase from Xanthomonas citri subsp. citri. The structure bears all the hallmarks of a dimeric member of the MnSOD family, including the conserved hydrogen-bonding network residues. Despite the apparent gene redundancy, several attempts to obtain a sodM deletion mutant were unsuccessful, suggesting the encoded protein to be essential for bacterial survival. This intriguing observation led us to extend our structural studies to the remaining three SOD homologs, for which comparative models were built. The models imply that X. citri subsp. citri produces an iron-containing SOD which is unlikely to be catalytically active along with two conventional Cu,ZnSODs. Although the latter are expected to possess catalytic activity, we propose they may not be able to replace XcSOD for reasons such as distinct subcellular compartmentalization or differential gene expression in pathogenicity-inducing conditions.


Assuntos
Proteínas de Bactérias/química , Superóxido Dismutase/química , Xanthomonas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Genes Essenciais , Modelos Moleculares , Conformação Proteica , Superóxido Dismutase/genética , Xanthomonas/genética , Xanthomonas/patogenicidade
6.
Mol Plant Pathol ; 19(1): 143-157, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798950

RESUMO

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.


Assuntos
Membrana Celular/metabolismo , Proteínas Periplásmicas/metabolismo , Proteômica , Xanthomonas/metabolismo , Xanthomonas/patogenicidade , Proteínas de Bactérias/metabolismo , Eletroforese em Gel Bidimensional , Modelos Biológicos , Proteoma/metabolismo
7.
J Proteomics ; 151: 251-263, 2017 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-27180281

RESUMO

Xanthomonas citri subsp. citri (XAC) is the causative agent of citrus canker, a disease of great economic impact around the world. Understanding the role of proteins on XAC cellular surface can provide new insights on pathogen-plant interaction. Surface proteome was performed in XAC grown in vivo (infectious) and in vitro (non-infectious) conditions, by labeling intact cells followed by cellular lysis and direct 2D-DIGE analysis. Seventy-nine differential spots were analyzed by mass spectrometry. Highest relative abundance for in vivo condition was observed for spots containing DnaK protein, 60kDa chaperonin, conserved hypothetical proteins, malate dehydrogenase, phosphomannose isomerase, and ferric enterobactin receptors. Elongation factor Tu, OmpA-related proteins, Oar proteins and some Ton-B dependent receptors were found in spots decreased in vivo. Some proteins identified on XAC's surface in infectious condition and predicted to be cytoplasmic, such as DnaK and 60KDa chaperonin, have also been previously found at cellular surface in other microorganisms. This is the first study on XAC surface proteome and results point to mediation of molecular chaperones in XAC-citrus interaction. The approach utilized here can be applied to other pathogen-host interaction systems and help to achieve new insights in bacterial pathogenicity toward promising targets of biotechnological interest. BIOLOGICAL SIGNIFICANCE: This research provides new insights for current knowledge of the Xanthomonas sp. pathogenicity. For the first time the 2D-DIGE approach was applied on intact cells to find surface proteins involved in the pathogen-plant interaction. Results point to the involvement of new surface/outer membrane proteins in the interaction between XAC and its citrus host and can provide potential targets of biotechnological interest for citrus canker control.


Assuntos
Citrus/microbiologia , Interações Hospedeiro-Patógeno , Proteoma/análise , Xanthomonas/patogenicidade , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/análise , Proteínas de Transporte/análise , Proteínas de Transporte/fisiologia , Proteínas de Membrana/análise , Proteínas de Membrana/fisiologia , Doenças das Plantas/microbiologia , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/fisiologia , Eletroforese em Gel Diferencial Bidimensional , Xanthomonas/química
8.
PLoS One ; 11(9): e0162886, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27611974

RESUMO

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a severe disease of citrus. Xcc presents broad spectrum of citrus hosts including economically important species whereas X. fuscans subsp. aurantifolii-type C (XauC) causes a milder disease and only infects Citrus aurantifolia. Trehalase catalyzes hydrolysis of the disaccharide trehalose, a sugar that has been reported to be related to Xcc pathogenicity. We expressed the recombinant gene product and assessed Xcc trehalase structural and kinetics data. The recombinant protein presented 42.7% of secondary structures in α-helix and 13% in ß-sheets, no quaternary structure in solution, and Michaelis-Menten constant (KM) of 0.077 mM and Vmax 55.308 µMol glucose.min-1.mg protein-1 for trehalose. A Xcc mutant strain (XccΔtreA) was produced by gene deletion from Xcc genome. Enzymatic activity of trehalase was determined in Xcc, XauC and XccΔtreA cellular lysates, showing the highest values for XauC in in vitro infective condition and no activity for XccΔtreA. Finally, leaves of Citrus aurantifolia infected with XccΔtreA showed much more drenching and necrosis than those infected by wild type Xcc. We concluded that trehalase contributes to alleviate bacterial virulence and that inability for trehalose hydrolysis may promote higher Xcc infectivity.


Assuntos
Genes Bacterianos , Trealase/metabolismo , Xanthomonas/enzimologia , Xanthomonas/patogenicidade , Cromatografia em Gel , Dicroísmo Circular , Citrus/microbiologia , Deleção de Genes , Cinética , Doenças das Plantas/microbiologia , Proteínas Recombinantes/metabolismo , Trealase/genética , Xanthomonas/química , Xanthomonas/genética
9.
Data Brief ; 8: 1400-11, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27595129

RESUMO

Here we provide the mass-spectrometry and in silico interaction network dataset of proteins identified on our research article on surface proteomic analysis from Xanthomonas citri subsp. citri (XAC) cells grown in vivo (infectious) and in vitro (non-infectious, control) by 2D-DIGE approach. Fluorescence labeling of proteins were performed on intact cells followed by cellular lysis and labeled spots from 2D gel differing in abundance between the two conditions (ANOVA, p-value<0.05) were analyzed by a nano-electrospray tandem mass spectrometry Q-Tof Ultima API mass spectrometer (MicroMass/Waters) (LC-ESI-MS/MS). This article contains raw data of proteins detected in the 79 spots analyzed by LC-ESI-MS/MS approach and also an enrichment analysis on the resulting protein-protein interaction network performed with the Integrated Interactome System (IIS) platform and Cytoscape software. The data are supplementary to our original research article, "Xanthomonas citri subsp. citri surface proteome by 2D-DIGE: ferric enterobactin receptor and other outer membrane proteins potentially involved in citric host interaction" (Carnielli et al., 2016) [1], and raw data are available via Peptide Atlas (ftp://PASS00850:ZJ7425v@ftp.peptideatlas.org/).

10.
Braz. j. microbiol ; 45(4): 1117-1129, Oct.-Dec. 2014. tab
Artigo em Inglês | LILACS | ID: lil-741261

RESUMO

Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.


Assuntos
Animais , Humanos , Vacinas Bacterianas/imunologia , Portadores de Fármacos , Infecções Bacterianas/prevenção & controle , Vacinas Bacterianas/genética , Neoplasias/terapia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia
11.
Braz J Microbiol ; 45(4): 1117-29, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25763014

RESUMO

Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.


Assuntos
Vacinas Bacterianas/imunologia , Portadores de Fármacos , Animais , Infecções Bacterianas/prevenção & controle , Vacinas Bacterianas/genética , Humanos , Neoplasias/terapia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia
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