Evidences of siderophores synthesis by Grapevine Xylella fastidiosa, causal agent of pierce's disease, through instrumental approaches

Abstract

Os sideróforos provenientes de Xylella fastidiosa de videiras foram investigados. Tais metabólitos seqüestram ferro, um elemento essencial, do hospedeiro, o que os torna um potencial fator de patogenicidade. Em um meio de cultura em placa com limitação de ferro, tais sideróforos foram detectados pela reação com o complexo cromoazurol S (CAS). Diferentes métodos de análise instrumentais foram utilizados para caracterização dos sideróforos, como: cromatografia de afinidade por metal imobilizado (IMAC), cromatografia micelar eletrocinética capilar (MEKC) e espectrometria de massas com ionização por electrospray (ESI-MS). Os resultados obtidos confirmaram a produção de sideróforos. A extração do(s) composto(s) por IMAC com Fe 3+ imobilizado foi uma etapa importante. O(s) sideróforo(s) não foi separado por eletroforese capilar de zona, indicando sua neutralidade sob os pHs investigados. As análises por MEKC apresentaram um pico diferente (quando comparadas à análise do controle), com caráter levemente hidrofóbico. A espectrometria de massas mostrou que os compostos alvos podem ter uma massa molecular relativa dentro da esperada para sideróforos, como: 875, 1004 e 1092 Da.Siderophore molecules from grapevine Xylella fastidiosa were investigated. Such metabolites sequester iron, an essential element, from the host, making them a potential factor of pathogenicty. In an iron-limited medium, siderophores were detected in culture plates of X. fastidiosa containing the complex Chromeazurol S (CAS). A combination of different instrumental analyses was used for siderophore(s) characterization, such as: immobilized metal affinity chromatography (IMAC), micelar electrokinetic capillary chromatography (MEKC) and electrospray-mass spectrometry (ESI-MS). The results show that grapevine X. fastidiosa produced siderophore(s), as confirmed by the CAS plate assay. The extraction of the compound(s) using IMAC with immobilized Fe 3+ was important for analyte purification. The chelator was not separated by capillary zone electrophoresis, indicating the possibility of a neutral compound under the investigated pHs. MEKC runs presented a different peak (when compared to the control analysis) which represented a slightly hydrophobic compound. Mass spectrometry showed that the compound(s) may have a relative molecular mass within the expected range for siderophore molecules, such as 875, 1004 and 1092 Da. Keywords: IMAC, MEKC, ESI-MS, siderophores, iron sequestration IntroductionXylella fastidiosa is a gram-negative pathogenic bacterium in grapevine, resulting in Pierce's disease, and in a large variety of other hosts. Many diseases are caused by X. fastidiosa, citrus variegated chlorosis (CVC) 3 and Pierces' disease of grapevine (PD) 2,3 being economically the most important ones. Other diseases caused by this phytopathogen are found in the following hosts: peach, plum, 5 coffee, 6 almond, sycamore, alfalfa, among others.Evidences of Siderophores Synthesis by Grapevine Xylella fastidiosa J. Braz. Chem. Soc. 636 CVC has been an econom...

Fe(III)-based immobilized metal–affinity chromatography (IMAC) method for the separation of the catechol siderophore from Bacillus tequilensis CD36

“…As shown in Fig. 5, the purified catechol siderophore DHB-Gly-Thr (M = 312), which is the single unit of bacillibactin, could be obtained by (Hart et al 2002;Barnouin et al 2005), and hydroxamate-containing siderophores [Ni(II)-IMAC, Fe(III)-IMAC] (Braich and Codd 2008;Simionato et al 2010;Ejje et al 2013). As bacillibactin is a secondary metabolite with low yield, and only the single unit of bacillibactin DHB-Gly-Thr was separated in this study.…”

“…When sample was loaded, the incoming active substances were bound to the immobilized metal and separated from the rest of sample. Thus, Fe 3+ -immobilized IMAC columns may be successfully used for the extraction of siderophore from complex samples (Simionato et al 2010). Najwa Braich and Rachel Codd had already successfully captured hydroxamate-containing siderophores and other Fe(III)-binding metabolites directly from bacterial culture supernatants with nickel(II)-based IMAC (Braich and Codd 2008), but there are no records of Fe(III)-based IMAC for the separation of catechol siderophore.…”

Metal-Chelating Peptides Separation Using Immobilized Metal Ion Affinity Chromatography: Experimental Methodology and Simulation

“…Between the matrix and the chelating agent, a short alkyl chain (the socalled spacer arm) is grafted to ensure accessibility. The IMAC column can be purchased already filled with the matrix grafted with the complexing agent [59,[83][84][85] or the matrix can be bought separately [86][87][88][89][90]. Inversely, an initially empty column can be filled with resins [15,57,91,92].…”

“…Metal ions are frequently loaded onto resins in their hydrochloride form [106][107][108] or as sulfates salts [15,90,91]. The metal ions loaded in excess are removed using either sodium chloride solutions, a weak complex agent such as acetate [59,109], a low concentration of a strong complexing agent (phosphate, <50 mM) [110,111], or simply with deionized water.…”

“…Unbound components are removed using the loading buffer or deionized water [15,88,[90][91][92]106,113]. When water is used for washing, it is followed by a second wash with a weak complexing agent such as formate or acetate [45,59,94,94,109,115].…”