Estudio de bioprospección de compuestos inhibidores de la comunicación celular (QS) como estrategia de control de agentes fitopatógenos

En esta tesis el aislamiento se hizo de manera bioguiada usando el biosensor Chromobacterium violaceum ATCC 31532. Los compuestos puros identificados también se evaluaron como inhibidores del QS (IQS) frente a Burkholderia glumae CIAT 4026, aislada de cultivos de arroz en Colombia, causante del añublo bacterial de la panícula de arroz. Algunos fenotipos de virulencia controlados por QS en B. glumae son la producción de toxoflavina y motilidad swimming\ Para evaluar el potencial metabólico de los microorganismos procedentes del Mar Caribe colombiano, se recolectaron muestras de invertebrados, algas, peces y sedimentos marinos. Se aislaron 203 microorganismos. Entre ellos 162 se describieron como bacterias, y 41 como hongos. El estudio empezó con bacterias del Phylum Actinobacteria (géneros Streptomyces, Micromonospora y Gordonia), reconocido como una fuente de compuestos con actividad biológica. Se identificaron 24 Actinobacterias, de las cuales 8 resultaron activas como IQS. Los extractos orgánicos fueron analizados por HPLC-MS, con esta información se llevó a cabo un estudio metabolómico que permitió relacionar las variables que describen al sistema con los valores de actividad biológica. Estos resultados permitieron identificar preliminarmente algunos compuestos activos como la streptomicina D. Finalmente se plantea una discusión sobre qué información usar (actividad biológica, perfil por LC-MS, información taxonómica) para la priorización de las cepas a estudiar, planteando un enfoque integrador y los posibles resultados de esta estrategia. Se eligieron dos cepas para realizar estudios químicos preliminares: Micromonospora sp. PNM102N, y Steptomyces sp. PNM89.3 por ser las que presentaban buena actividad biológica. De estas cepas se aisló compuestos lipídicos como los responsables de la actividad. No se continuó con el estudio químico de estos compuestos porque se recuperan en muy baja cantidad y su actividad no es muy fuerte. De los 138 aislamientos que no pertenecen al Phylum Actinobacteria se idenficaron 8 cepas activas. Estas fueron caracterizadas bioquímicamente e identificadas molecularmente correspondiendo a los géneros: Bacillus, Paenibacillus, Lisynibacillus y Proteus. Una no pudo ser caracterizada molecularmente. Para el estudio químico se seleccionó a la cepa PNM115 (Paenibacillus sp), por ser la más activa y presentar su actividad en la fase butanólica. Un estudio detallado de los espectros mono y bi dimensionales llevaron a caracterizarlo como un péptido análogo de las polimixinas con unidades Val-Ser, Asn-Phe, ácido léucico, leucina, isoleucina, dos unidades de cisteína y una de ácido 3-hidroxi-4-metilhexanoico, y una masa de 1119.7135. Además, se buscó determinar si los compuestos volátiles (COVs) producidos por los aislamientos eran los responsables de la actividad IQS. Para esto las 16 cepas fueron evaluadas en un ensayo que asegura que el contacto a través del espacio de cabeza. Se encontró que sólo, la cepa PNM216 mostró actividad, ésta aún no ha sido identificada molecularmente. Del estudio por SPME y CG-EM se identificaron 29 compuestos diferentes a los producidos por el medio de cultivo. De éstos, 13 se ensayaron frente al biosensor. Así se logró establecer que cuatro compuestos: p-cimeno, el cumeno, el 5-metil-2-hexanona y la 2-nonanona eran activos inhibiendo la violaceina. Para determinar la efectividad de los compuestos como IQS se hicieron curvas de crecimiento en el que se encontró que tres parecían actuar como bacteriostáticos y el compuesto 5-metil-2-hexanona no afectaba el crecimiento, por lo tanto, es QQ. Se evaluaron otros compuestos volátiles, no identificados en el espacio de cabeza de la bacteria, entre estos fueron activos el acetato de isoamilo y el alcohol isoamílico. El estudio se continuó con la exploración de otras fuentes como esponjas del orden Haplosclerida, recolectadas en el Caribe colombiano. Se recolectaron cinco esponjas de los géneros Haliclona y Amphimedon. De estas únicamente Amphimedon viridis demostró actividad en la fracción butanólica de la cual se aisló el complejo halitoxina. De la fracción WW, también activa, se aisló la taurina. Finalmente, se estudiaron 7 especies de plantas del subgénero Tacosnia y una del subgénero Passiflora (Passifloraceae) Se realizó el perfilado metabolómico, por RMN 1H, lográndose detectar 53 compuestos de diferente naturaleza química. Los datos de RMN-1H para los extractos fueron correlacionados con su actividad QQ mediante un análisis supervisado OPLS-DA, identificando que los compuestos responsables de la actividad eran los flavonoides de P. lehmannii y P. uribei, que además son nuevos. Para evaluar la efectividad in vivo de los compuestos, se usó un modelo con catáfilos de cebolla infectados con el fitopatógeno del arroz B. glumae. Este modelo permite predecir los resultados a obtener con semillas de arroz.Abstract: Quorum sensing (QS) is a bacterial cell-cell communication process, which allow s the regulation of gene expression in response to fluctuations in cell-population density. It is recognized that the quorum sensing inhibition can lead to prevent bacteria expression of virulence factors, essential for pathogen colonization or disease development along their interaction with the host. Therefore, the aim of this study was to contribute to control bacterial phytopathogens through the exploration of natural products as quorum sensing inhibitors. The role of quorum quenchers (QQ) to control bacterial phytopathogens has been poorly, it has become necessary to search for compounds with quorum quenching activity. In this study, screening of QS inhibitors were performed over several natural sources such as bacteria, sponges and plants. This guaranteed a wide exploration of the chemical space, including compounds of low and high polarity, volatiles and nonvolatilesDoctorad

Passiflora

<i>2.3. Bioprospecting studies of Passiflora species related to QQ activity</i> <p> Having completed the metabolic profiling of the studied <i>Passiflora</i> species, the next step was to relate those profiles with the biological activity observed for the extracts in order to identify the compounds responsible for such an activity (Wu et al., 2015). The selected bioactivity was quorum sensing inhibitory activity (QSI activity) because the search of anti-pathogenic compounds seemed to be a better strategy than the search for antibiotics, in terms of reducing the damage in the host, without generating induced resistance in the pathogen. Several small molecules including <i>C</i> -glycoside flavonoids, vanillin, 3-indolyacetonitrile, among others have been reported to be quorum sensing inhibitors (Grandclément et al., 2016), (Brango-Vanegas et al., 2014).</p> <p> The MeOH/H 2 O extracts of <i>Passiflora</i> species were tested for the inhibition of violacein production using <i>Chromobacterium violaceum</i> ATCC 31532 as a biosensor (supporting info table 4). Results showed that <i>P. uribei</i>, <i>P. lehmannii</i> and <i>P. cumbalensis</i> exerted a strong activity (inhibition halo> 40 mm) (Fig. 17 supporting information) while other <i>Passiflora</i> samples showed less or no activity at all. The complete results are summarized in Supp. Table 4.</p> <p> The metabolites that had been detected by 1 H-NMR were correlated with the bioactivity (QSI) by applying the orthogonal projection to latent structures (OPLS-DA), using the coded QSI activity (20 mm-inhibition zone was coded as 1;> 30 mm of inhibition zone was coded as 3) as the Y-variable. Separation of the active groups is observed in the OPLS-DA score plot (R 2 = 0.425 and Q 2 = 0.302, pareto scaling), with the active groups on the negative side along OPLS1 (Fig. 7A). <i>Passiflora cumbalensis</i> clustered as a well-defined active group, while the other species did not show a clear clustering tendency. Three active groups were identified along the OPLS2 axis, one being on the negative side for <i>P. lehmannii</i> and <i>P. uribei,</i> one on the positive side for <i>P. cumbalensis</i> and a third one for the other species spread out in the middle of the plot, suggesting that the active compounds for these three groups were different.</p> <p> Using two <i>S-plots</i>, one excluding <i>P. lehmanii</i> samples (Fig. 7B) and the other excluding <i>P. cumbalensis</i> samples (Fig. 7C) it was possible to identify the active compounds. The variables important for the projection (VIPs) were selected, and the chemical shifts responsible for the QSI activity were highlighted. These highlighted chemical shifts were found to correspond mostly to the glycosylated flavonoids because the signals could be assigned to aromatic protons such as those of the A and B rings from flavonoids as well as signals for sugar moieties, including those of the anomeric protons close to 5 ppm (Tables 5 and 6, Supporting info).</p> <p> The quality and robustness of the OPLS-DA model was validated by a permutation test (n = 100). The Q 2 intercept value was −0.504 (below 0.05), showing that the original model was statistically effective (Fig. 18 Supporting info). The model was validated by calculating the area under the receiver operating characteristic (ROC) curve. The value of the area under the curve (AUC) was 0.9565 providing added confidence to the model (Fig. 18B supporting info).</p> <p> Pure compounds <b>1</b> and <b>2</b> were tested for their QS inhibition against <i>C. violaceum</i> at five concentrations in the range of 50 μM–400 μM in a 96 well-plate. The QS inhibition of compound <b>1</b> and compound <b>2</b> was detected at concentrations of 100 μg/mL (0.13 mM) and 300 μg/mL (0.47 mM) respectively. In order to establish whether the observed inhibition was due solely to QS inhibition and not to growth inhibition, samples were submitted to a growth inhibition test (Fig. 19 supporting information). Results of the assays showed not only the absence of growth inhibition but an increase in bacterial cell densities, indicating that the flavonoids likely inhibited cell communication.</p> <p> A second model, <i>Burkholderia glumae</i>, a well-known phytopathogen that causes rice grain rot and wilt in various field crops was also used to evaluate QSI (Compant et al., 2008). In <i>B. glumae</i>, the production of toxoflavin (a bright yellow pigment) is known to be one of the major virulence factors (Jeong et al., 2003; J. Kim et al., 2004). The biosynthesis of toxoflavin is controlled by ToxR, a LysR-type transcriptional regulator and this toxin also activates the expression of the <i>tox</i> operons (J. Kim et al., 2004). For this reason, the search for compounds that are able to inhibit toxoflavin production is an important target for the control of this phytopathogen. Two strains were chosen to determine the toxoflavin inhibitory activity of extracts and pure compounds. <i>Burkholderia glumae</i> COK 71, is a biosensor strain, that is highly specific for toxoflavin based on β- galactosidase activity on X gal substrate that produces a blue pigment, and the <i>B. glumae</i> ATCC 33617 strain as a toxoflavin producer. In this test, the levels of the blue pigment are used to determine toxoflavin inhibitory activity (Choi et al., 2013). Our results indicated that toxoflavin productions was inhibited by concentrations of 6.76 μM and 7.87 μM of compounds <b>1</b> and <b>2,</b> respectively, while the positive control, 2- <i>n</i> -propyl-9-hydroxy-4H-pyrid [1,2-a] pyrimidin-4-one was active at 80 μM, showing the potential of these flavonoids to control toxin production by the phytopathogen, <i>B. glumae</i> (Fig. 20, supporting information).</p> <p> The presence of flavonoids in plant extracts has been previously related to their QS inhibition activity. Phytochemical screening of <i>Centella asiatica</i> has revealed that flavonoids can disrupt AHL-mediated QS-controlled systems in <i>C. violaceum</i> and <i>P. aeruginosa</i> while major constituents such as the triterpene, asiatic acid, did not show an anti-QS activity (Vasavi et al., 2016). Concentrations of 100 μg/mL of quercetin and kaempferol have been reported to exhibit anti-QS activity against <i>C. violaceum</i> and <i>P. aeruginosa</i> PAO 1. The anti-QS activity of <i>Psidium guajava</i> leaf extract has been determined with a biosensor bioassay using <i>Chromobacterium violaceum</i> CV 026, and quercetin and quercetin 3- <i>O</i> -arabinoside were identified as the QQ compounds in the extract, against <i>C. violaceum</i> 12,472, at concentrations of 50 and 100 μg/mL, respectively (Vasavi et al., 2014). Similarly, Paczkowski et al. studied the QS inhibition mechanism of flavonoids, establishing that they are inhibitors of the QS transcriptional regulator LasR and that they specifically inhibit quorum sensing via antagonism with the transcriptional regulator LasR/RhlR. Further structure-activity relationship analyses suggest that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses also revealed that flavonoids function non-competitively to prevent LasR/RhlR DNA-binding. The administration of the flavonoids to <i>P. aeruginosa</i> was found to alter transcription of the quorum-sensing controlled target promoters and suppress virulence factor production, confirming their potential as antimicrobials which do not function by traditional bactericidal or bacteriostatic mechanisms (Paczkowski et al., 2017).</p>Published as part of <i>Castellanos, Leonardo, Naranjo-Gaybor, Sandra Judith, Forero, Abel M., Morales, Gustavo, Wilson, Erica Georgina, Ramos, Freddy A. & Choi, Young Hae, 2020, Metabolic fingerprinting of banana passion fruits and its correlation with quorum quenching activity, pp. 1-13 in Phytochemistry (112272) (112272) 172</i> on pages 8-9, DOI: 10.1016/j.phytochem.2020.112272, <a href="http://zenodo.org/record/8294125">http://zenodo.org/record/8294125</a&gt