RESUMO
This study aims to obtain secondary metabolites extracts from filamentous fungi isolated from soil and marine sediments from Antarctic ecosystems and to assess its potential antibacterial activity on Xanthomonas euvesicatoria and Xanthomonas axonopodis pv. passiflorae (phytopathogenic bacteria causing diseases in pepper and tomato and passionfruit, respectively). Among the 66 crude intracellular and extracellular extracts obtained from fungi recovered from soil and 79 obtained from marine sediment samples, 25 showed the ability to prevent the growth of X. euvesicatoria in vitro and 28 showed the ability to prevent the growth of X. axonopodis pv. passiflorae in vitro. Intracellular and extracellular extracts from soil fungi inhibited around 97% of X. euvesicatoria and 98% of X. axonopodis pv. passiflorae at 2·1 mg ml-1 . The average inhibition rates against X. euvesicatoria and X. axonopodis pv. passiflorae for intracellular and extracellular extracts from marine sediments fungi were around 96 and 97%, respectively, at 3·0 mg ml-1 . Extracts containing secondary metabolites with antimicrobial activity against X. euvesicatoria and X. axonopodis pv. passiflorae were obtained, containing possible substitutes for the products currently used to control these phytopathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Micro-organisms from extreme ecosystems, such as the Antarctic ecosystem, need to survive in harsh conditions with low temperatures, low nutrients and high UV radiation. Micro-organisms adapt to these conditions evolving diverse biochemical and physiological adaptations essential for survival. All this makes these micro-organisms a rich source of novel natural products based on unique chemical scaffolds. Discovering novel bioactive compounds is essential because of the rise in antibiotic-resistant micro-organisms and the emergence of new infections. Fungi from Antarctic environments have been proven to produce bioactive secondary metabolites against various micro-organisms, but few studies have shown activity against Xanthomonas phytopathogens.
Assuntos
Antibacterianos/farmacologia , Capsicum/microbiologia , Extratos Celulares/farmacologia , Fungos/metabolismo , Passiflora/microbiologia , Doenças das Plantas/microbiologia , Solanum lycopersicum/microbiologia , Xanthomonas axonopodis/efeitos dos fármacos , Regiões Antárticas , Fungos/química , Sedimentos Geológicos/microbiologia , Microbiologia do Solo , Xanthomonas axonopodis/isolamento & purificaçãoRESUMO
BACKGROUND: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). METHODOLOGY/PRINCIPAL FINDINGS: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. CONCLUSIONS: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.
Assuntos
Catalase/metabolismo , Citrus/microbiologia , Xanthomonas axonopodis/enzimologia , Xanthomonas axonopodis/patogenicidade , Adaptação Fisiológica/efeitos dos fármacos , Catalase/biossíntese , Catalase/genética , Meios de Cultura , Farmacorresistência Bacteriana/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/microbiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação/genética , Virulência/efeitos dos fármacos , Xanthomonas axonopodis/efeitos dos fármacos , Xanthomonas axonopodis/crescimento & desenvolvimentoRESUMO
The most severe form of citrus canker disease is caused by Xanthomonas axonopodis pv. citri (Xac) and affects all types of important citrus crops, reducing fruit yield and quality. Copper-based products are routinely used as a standard control measure for citrus canker. In this work we demonstrate that copper treatment induces the viable but nonculturable (VBNC) state in Xac but does not prevent the development of symptoms in susceptible plants. Short-term exposures to different concentrations of copper solutions were assayed to determine which treatment resulted in Xac nonculturability. Treatment of 10(6) mL(-1) Xac cells for 10 min in a 135-muM CuSO(4) solution (equivalent to three times the free soluble copper concentration applied in one field treatment) resulted in nonculturability. However, 16% of cells were viable based on 5-cyano-2,3-ditolyl tetrazolium chloride staining and 1% were capable of producing canker lesions after infiltrating grapefruit plants. If induction of the VBNC state in Xac cells were to occur under field conditions, this would have to be taken into consideration for an effective control of canker disease.
Assuntos
Antibacterianos/farmacologia , Sulfato de Cobre/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Xanthomonas axonopodis/efeitos dos fármacos , Xanthomonas axonopodis/patogenicidade , Citrus paradisi/microbiologia , Doenças das Plantas/microbiologia , Virulência/efeitos dos fármacosRESUMO
This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its V(max) (7.56nmoles/min) and K(M) (2.76mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75mM and 0.4muM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2.