RESUMO
Streptomyces scabies is responsible for common scab disease on root and tuber vegetables. Production of its main phytotoxin thaxtomin A is triggered upon transport of cellulose byproducts cellotriose and cellobiose, which disable the repression of the thaxtomin biosynthesis activator gene txtR by the cellulose utilization regulator CebR. To assess the intracellular response under conditions where S. scabies develops a virulent behavior, we performed a comparative proteomic analysis of wild-type S. scabies 87-22 and its cebR null mutant (hyper-virulent phenotype) grown in the absence or presence of cellobiose. Our study revealed significant changes in abundance of proteins belonging to metabolic pathways known or predicted to be involved in pathogenicity of S. scabies. Among these, we identified proteins of the cello-oligosaccharide-mediated induction of thaxtomin production, the starch utilization system required for utilization of the carbohydrate stored in S. scabies's hosts, and siderophore synthesis utilization systems, which are key features of pathogens to acquire iron once they colonized the host. Thus, proteomic analysis supported by targeted mass spectrometry-based metabolite quantitative analysis revealed the central role of CebR as a regulator of virulence of S. scabies.
Assuntos
Proteínas de Bactérias/genética , Celobiose/farmacologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Indóis/metabolismo , Piperazinas/metabolismo , Streptomyces/genética , Proteínas de Bactérias/metabolismo , Celobiose/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Eletroforese em Gel Bidimensional , Ontologia Genética , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Doenças das Plantas/microbiologia , Proteômica/métodos , Sideróforos/biossíntese , Sideróforos/isolamento & purificação , Streptomyces/efeitos dos fármacos , Streptomyces/metabolismo , Streptomyces/patogenicidade , Espectrometria de Massas em Tandem , VirulênciaRESUMO
RATIONALE: Burkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking. METHODS: Based on results of a label-free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors. RESULTS: Increase in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non-physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF. CONCLUSIONS: Microfluidics-based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens.
RESUMO
The family Anisakidae, mainly represented by Anisakis simplex s.l. and Pseudoterranova decipiens, encompasses zoonotic nematodes infecting many marine fish. Both are responsible for gastrointestinal disease in humans after ingestion of a live larva by consumption of undercooked fish, and, in the case of A. simplex, an allergic reaction may occur after consuming or even handling infected fish. Due to its phylogenetic relatedness with A. simplex, few studies investigated the allergenic potential of P. decipiens, yet none of them focused on its excretory/secretory (E/S) proteins that easily get missed when working solely on extracts from crushed nematodes. Moreover, these E/S allergens remain behind even when the larva has been removed during fish quality processing. Therefore, the aim was to investigate if Anisakis-like allergens could also be detected in both crushed and E/S P. decipiens protein extract using targeted mass spectrometry analysis and immunological methods. The results confirmed that at least five A. simplex allergens have homologous proteins in P. decipiens; a result that emphasizes the importance of also including E/S protein extracts in proteomic studies. Not only A. simplex, but also P. decipiens should therefore be considered a potential source of allergens that could lead to hypersensitivity reactions in humans.
Assuntos
Anisakis , Ascaridoidea , Hipersensibilidade , Alérgenos , Animais , Peixes , Imunoensaio , Larva/metabolismo , Filogenia , Proteômica/métodosRESUMO
The development of spots or lesions symptomatic of common scab on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or a transcriptional response to cello-oligosaccharides. Comparative metabolomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include (i) thaxtomin and concanamycin phytotoxins, (ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, (iii) ectoine for protection against osmotic shock once inside the host, and (iv) bottromycin and concanamycin antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cello-oligosaccharides reduced the production of the spore germination inhibitors germicidins thereby giving the 'green light' to escape dormancy and trigger the onset of the pathogenic lifestyle. For most metabolites - either with induced or reduced production - cellotriose was revealed to be a slightly stronger elicitor compared to cellobiose, supporting an earlier hypothesis which suggested the trisaccharide was the real trigger for virulence released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs' expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of a yet unknown mechanism for switching on the virulome. Finally, a transcriptomic analysis revealed nine additional cryptic BGCs that have their expression awakened by cello-oligosaccharides, suggesting that other and yet to be discovered metabolites could be part of the virulome of S. scabiei.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Celobiose/farmacologia , Celulose/farmacologia , Tubérculos/microbiologia , Streptomyces/crescimento & desenvolvimento , Trioses/farmacologia , Fatores de Virulência/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Macrolídeos/metabolismo , Metabolômica , Família Multigênica/efeitos dos fármacos , Piperazinas/metabolismo , Tubérculos/crescimento & desenvolvimento , RNA-Seq , Streptomyces/efeitos dos fármacos , Streptomyces/metabolismo , Streptomyces/patogenicidadeRESUMO
Cellulose being the most abundant polysaccharide on earth, beta-glucosidases hydrolyzing cello-oligosaccharides are key enzymes to fuel glycolysis in microorganisms developing on plant material. In Streptomyces scabiei, the causative agent of common scab in root and tuber crops, a genetic compensation phenomenon safeguards the loss of the gene encoding the cello-oligosaccharide hydrolase BglC by awakening the expression of alternative beta-glucosidases. Here, we revealed that the BglC compensating enzyme BcpE2 was the GH3-family beta-glucosidase that displayed the highest reported substrate promiscuity and was able to release the glucose moiety of all tested types of plant-derived heterosides (aryl ß-glucosides, monolignol glucosides, cyanogenic glucosides, anthocyanosides, and coumarin heterosides). BcpE2 structure analysis highlighted a large cavity in the PA14 domain that covered the active site, and the high flexibility of this domain would allow proper adjustment of this cavity for disparate heterosides. The exceptional substrate promiscuity of BcpE2 provides microorganisms a versatile tool for scavenging glucose from plant-derived nutrients that widely vary in size and structure. Importantly, scopolin was the only substrate commonly hydrolyzed by both BglC and BcpE2, thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes would also fine-tune the strength of virulence. IMPORTANCE Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides. In this work, structural, enzymatic, and expression studies revealed the existence of a "swiss-army knife" enzyme, BcpE2, that was able to retrieve the glucose moiety of a multitude of plant-derived substrates that vary in size, structure, and origin. This enzyme would provide the microorganisms with a tool that would allow them to find nutrients from any type of plant-derived material.
Assuntos
Glucose , beta-Glucosidase , Glucose/metabolismo , Glucosídeos/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/metabolismoRESUMO
Streptomyces scabies is a phytopathogen associated with common scab disease. This is mainly attributed to its ability to produce the phytotoxin thaxtomin A, the biosynthesis of which is triggered by cellobiose. During a survey of other metabolites released in the presence of cellobiose, we discovered additional compounds in the thaxtomin-containing extract from Streptomyces scabies. Structural analysis by mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that these compounds are amino acid sequence variants of the TOR (target of rapamycin) kinase (TORK) pathway-inhibitory lipopeptide rotihibin A, and the main compounds were named rotihibins C and D. In contrast to thaxtomin, the production of rotihibins C and D was also elicited in the presence of glucose, indicating different regulation of their biosynthesis. Through a combination of shotgun and targeted proteomics, the putative rotihibin biosynthetic gene cluster rth was identified in the publicly available genome of S. scabies 87-22. This cluster spans 33 kbp and encodes 2 different nonribosomal peptide synthetases (NRPSs) and 12 additional enzymes. Homologous rth biosynthetic gene clusters were found in other publicly available and complete actinomycete genomes. Rotihibins C and D display herbicidal activity against Lemna minor and Arabidopsis thaliana at low concentrations, shown by monitoring the effects on growth and the maximal photochemistry efficiency of photosystem II. IMPORTANCE Rotihibins A and B are plant growth inhibitors acting on the TORK pathway. We report the isolation and characterization of new sequence analogues of rotihibin from Streptomyces scabies, a major cause of common scab in potato and other tuber and root vegetables. By combining proteomics data with genomic analysis, we found a cryptic biosynthetic gene cluster coding for enzyme machinery capable of rotihibin production. This work may lead to the biotechnological production of variants of this lipopeptide to investigate the exact mechanism by which it can target the plant TORK pathway in Arabidopsis thaliana. In addition, bioinformatics revealed the existence of other variants in plant-associated Streptomyces strains, both pathogenic and nonpathogenic species, raising new questions about the actual function of this lipopeptide. The discovery of a module in the nonribosomal peptide synthetase (NRPS) that incorporates the unusual citrulline residue may improve the prediction of peptides encoded by cryptic NRPS gene clusters.
Assuntos
Proteínas de Bactérias/genética , Herbicidas/metabolismo , Família Multigênica , Oligopeptídeos/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Celobiose/metabolismo , Regulação Bacteriana da Expressão Gênica , Herbicidas/farmacologia , Oligopeptídeos/farmacologia , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismoRESUMO
In the plant pathogen Streptomyces scabies, the gene bglC encodes a GH1 family cellobiose beta-glucosidase that is both required for primary metabolism and for inducing virulence of the bacterium. Deletion of bglC (strain ΔbglC) surprisingly resulted in the augmentation of the global beta-glucosidase activity of S. scabies. This paradoxical phenotype is highly robust as it has been observed in all bglC deletion mutants independently generated, thereby highlighting a phenomenon of genetic compensation. Comparative proteomics allowed to identify two glycosyl hydrolases - named BcpE1 and BcpE2 - of which peptide levels were significantly increased in strain ΔbglC. Quantitative RT-PCR revealed that the higher abundance of BcpE1 and BcpE2 is triggered at the transcriptional level, the expression of their respective gene being 100 and 15 times upregulated. Enzymatic studies with pure BcpE proteins showed that they both possess beta-glucosidase activity thereby explaining the genotypic-phenotypic discrepancy of the bglC deletion mutant. The GH1 family BcpE1 could hydrolyze cellobiose and generate glucose similarly to BglC itself thereby mainly contributing to the survival of strain ΔbglC when cellobiose is provided as sole nutrient source. The low affinity of BcpE2 for cellobiose suggests that this GH3 family beta-glucosidase would instead primarily target another and yet unknown glucose-beta-1,4-linked substrate. These results make S. scabies a new model system to study genetic compensation. Discovering how, either the bglC DNA locus, its mRNA, the BglC protein, or either its enzymatic activity controls bcpE genes' expression, will unveil new mechanisms directing transcriptional repression.
Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Fenótipo , beta-Glucosidase/genética , Proteínas de Bactérias/metabolismo , Ativação Enzimática , Isoenzimas , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Ativação Transcricional , beta-Glucosidase/química , beta-Glucosidase/metabolismoRESUMO
Common scab disease on root and tuber plants is caused by Streptomyces scabies and related species which use the cellulose synthase inhibitor thaxtomin A as the main phytotoxin. Thaxtomin production is primarily triggered by the import of cello-oligosaccharides. Once inside the cell, the fate of the cello-oligosaccharides is dichotomized: (i) the fuelling of glycolysis with glucose for the saprophytic lifestyle through the action of ß-glucosidase(s) (BGs); and (ii) elicitation of the pathogenic lifestyle by the inhibition of CebR-mediated transcriptional repression of thaxtomin biosynthetic genes. Here, we investigated the role of scab57721, encoding a putative BG (BglC), in the onset of the pathogenicity of S. scabies. Enzymatic assays showed that BglC was able to release glucose from cellobiose, cellotriose and all other cello-oligosaccharides tested. Its inactivation resulted in a phenotype opposite to that expected, as reduced production of thaxtomin was monitored when the mutant was cultivated on medium containing cello-oligosaccharides as unique carbon source. This unexpected phenotype could be attributed to the highly increased activity of alternative intracellular BGs, probably as a compensation for bglC inactivation, which then prevented cellobiose and cellotriose accumulation to reduce the activity of CebR. In contrast, when the bglC null mutant was cultivated on medium devoid of cello-oligosaccharides, it instead constitutively produced thaxtomin. This observed hypervirulent phenotype does not fit with the proposed model of the cello-oligosaccharide-mediated induction of thaxtomin production, and suggests that the role of BglC in the route to the pathogenic lifestyle of S. scabies is more complex than currently presented.
Assuntos
Streptomyces/patogenicidade , beta-Glucosidase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , beta-Glucosidase/genéticaRESUMO
Streptomyces scabies is an economically important plant pathogen well-known for damaging root and tuber crops by causing scab lesions. Thaxtomin A is the main causative agent responsible for the pathogenicity of S. scabies and cello-oligosaccharides are environmental triggers that induce the production of this phytotoxin. How cello-oligosaccharides are sensed or transported in order to induce the virulent behavior of S. scabies? Here we report that the cellobiose and cellotriose binding protein CebE, and MsiK, the ATPase providing energy for carbohydrates transport, are the protagonists of the cello-oligosaccharide mediated induction of thaxtomin production in S. scabies. Our work provides the first example where the transport and not the sensing of major constituents of the plant host is the central mechanism associated with virulence of the pathogen. Our results allow to draw a complete pathway from signal transport to phytotoxin production where each step of the cascade is controlled by CebR, the cellulose utilization regulator. We propose the high affinity of CebE to cellotriose as possible adaptation of S. scabies to colonize expanding plant tissue. Our work further highlights how genes associated with primary metabolism in nonpathogenic Streptomyces species have been recruited as basic elements of virulence in plant pathogenic species.