Anti-Quorum Sensing Agents from South Florida Medicinal Plants and their Attenuation of Pseudomonas Aeruginosa Pathogenicity

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

SILVER NANOPARTICLES AND THE PLANT-ASSOCIATING ABILITIES OF RHIZOBIACEAE BACTERIA

The use of nanoparticle technology in consumer products has been increasing due to their broad-spectrum antimicrobial properties. Specifically, silver nanoparticles (AgNPs) can demonstrate distinct physiochemical properties compared to bulk silver, including a large surface area to volume ratio that allows for higher reactivity with bacterial cell surfaces. AgNPs are being released into the environment, including soil ecosystems through various pathways such as points of production or during disposal of silver-containing products. This raises the concern about the potential impact on beneficial soil bacteria and their surrounding ecosystems. Members of the Rhizobiaceae family play important roles in nutrient cycling and contribute to overall soil fertility and the experiments in this thesis address the potential for AgNP-mediated toxicity on these plant-associating bacteria. Respiration analysis of Bradyrhizobium japonicum, Azospirillum brasilense, and Agrobacterium tumefaciens has revealed that AgNPs can negatively impact the growth and survival of these bacterial species, with B. japonicum being the most susceptible. Additionally, swimming motility assays using B. japonicum showed a significant decrease in colony diameter when treated with AgNPs (50 ppm). A significant decrease in root colonization of Triticum aestivum roots by A. brasilense was observed as AgNP treatment concentrations increased. Although some of the experiments could not be completed, taken together, these experiments and the research reported herein highlights the potential toxicological effects of AgNPs on bacterial species vital to the growth and health of agriculturally important crops.

Glutathione Transferases as Detoxification Agents: Structural and Functional Studies

Glutathione transferases (GSTs) are a diverse family of enzymes that catalyze the glutathione-dependent detoxification of toxic compounds. GSTs are responsible for the conjugation of the tripeptide glutathione (GSH) to a wide range of electrophilic substrates. These include industrial pollutants, drugs, genotoxic carcinogen metabolites, antibiotics, insecticides and herbicides. In light of applications in biomedicine and biotechnology as cellular detoxification agents, detailed structural and functional studies of GSTs are required. Plant tau class GSTs play crucial catalytic and non-catalytic roles in cellular xenobiotic detoxification process in agronomically important crops. The abundant existence of GSTs in Glycine max and their ability to provide resistance to abiotic and biotic stresses such as herbicide tolerance is of great interest in agriculture because they provide effective and suitable tools for selective weed control. Structural and catalytic studies on tau class GST isoenzymes from Glycine max (GmGSTU10-10, GmGSTU chimeric clone 14 (Sh14), and GmGSTU2-2) were performed. Crystal structures of GmGSTU10-10 in complex with glutathione sulfenic acid (GSOH) and Sh14 in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were determined by molecular replacement at 1.6 Å and 1.75 Å, respectively. Major structural variations that affect substrate recognition and catalytic mechanism were revealed in the upper part of helix H4 and helix H9 of GmGSTU10-10. Structural analysis of Sh14 showed that the Trp114Cys point mutation is responsible for the enhanced catalytic activity of the enzyme. Furthermore, two salt bridges that trigger an allosteric effect between the H-sites were identified at the dimer interface between Glu66 and Lys104. The 3D structure of GmGSTU2-2 was predicted using homology modeling. Structural and phylogenetic analysis suggested GmGSTU2-2 shares residues that are crucial for the catalytic activity of other tau class GSTs–Phe10, Trp11, Ser13, Arg20, Tyr30, Leu37, Lys40, Lys53, Ile54, Glu66 and Ser67. This indicates that the catalytic and ligand binding site in GmGSTU2-2 are well-conserved. Nevertheless, at the ligandin binding site a significant variation was observed. Tyr32 is replaced by Ser32 in GmGSTU2-2 and thismay affect the ligand recognition and binding properties of GmGSTU2-2. Moreover, docking studies revealed important amino acid residues in the hydrophobic binding site that can affect the substrate specificity of the enzyme. Phe10, Pro12, Phe15, Leu37, Phe107, Trp114, Trp163, Phe208, Ile212, and Phe216 could form the hydrophobic ligand binding site and bind fluorodifen. Additionally, side chains of Arg111 and Lys215 could stabilize the binding through hydrogen bonds with the –NO2 groups of fluorodifen. GST gene family from the pathogenic soil bacterium Agrobacterium tumefaciens C58 was characterized and eight GST-like proteins in A. tumefaciens (AtuGSTs) were identified. Phylogenetic analysis revealed that four members of AtuGSTs belong to a previously recognized bacterial beta GST class and one member to theta class. Nevertheless, three AtuGSTs do not belong to any previously known GST classes. The 3D structures of AtuGSTs were predicted using homology modeling. Comparative structural and sequence analysis of the AtuGSTs showed local sequence and structural characteristics between different GST isoenzymes and classes. Interactions at the G-site are conserved, however, significant variations were seen at the active site and the H5b helix at the C-terminal domain. H5b contributes to the formation of the hydrophobic ligand binding site and is responsible for recognition of the electrophilic moiety of the xenobiotic. It is noted that the position of H5b varies among models, thus providing different specificities. Moreover, AtuGSTs appear to form functional dimers through diverse modes. AtuGST1, AtuGST3, AtuGST4 and AtuGST8 use hydrophobic ‘lock–and–key’-like motifs whereas the dimer interface of AtuGST2, AtuGST5, AtuGST6 and AtuGST7 is dominated by polar interactions. These results suggested that AtuGSTs could be involved in a broad range of biological functions including stress tolerance and detoxification of toxic compounds.

Caracterização funcional de dois cDNAs homólogos à ciclofilina e à proteína reprimida por auxina (ARP) identificados em bibliotecas subtrativas a para floração em tomateiro

Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants.

Nuevas perspectivas sobre la histopatología de la infección de Pinus Radiata por Fusarium Circinatum

223 p.

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

Estudo da associação dos genes GGR, GGPPS2 e GGPPS6 em Arabidopsis thaliana L. (Brassicaceae) a compostos alelopáticos

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016.

Influência de diferentes meios de cultura na transformação genética de milho tropical.

2016