Responses of Myxobacteria to Plant-Related Chemosignals Suggests Their Roles in the Rhizobiome

In the rhizosphere, plants curate and maintain distinct rhizobiome, some of which serve as secondary defense mechanisms against pathogenic microorganisms. Additionally, in response to biotic stress, plants generate phytohormones which regulate signaling pathways to activate systemic resistance. These phytohormones contribute to the chemical space within the rhizosphere in addition to other low-molecular-weight metabolites used for cell-to-cell communication within species and across kingdoms. However, little is known about the influences of stress-related phytohormones on beneficial bacteria even though biocontrol strategies abundantly explore plant-beneficial microorganisms to manage pathogens in agricultural systems. Myxobacteria are competent predators of plant pathogens and have demonstrated responses when exposed to exogenous quorum signals produced by prey bacteria. Our objective was to study the impacts of exogenous phytohormones and plant-related signals on myxobacterial motility, global transcriptome, and metabolism to reveal the potential roles of these bacteria in the rhizosphere. The plant-associated myxobacterium Archangium sp. strain Cb G35 exhibited a p \u3c 0.05 increase in motility on exposure to methyljasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA) while transcriptomic studies revealed a ? fourfold change (p \u3c 0.05) in the transcription for 56 genes in response to MeJA exposure. Untargeted analysis of LC-MS/MS datasets of crude extracts from exposure experiments with Global Natural Products Social Molecular Networking (GNPS) and XCMS-MRM tools further highlighted the activation and deactivation of specialized metabolites in response to these signals. Antibacterial assays of fractions from active extracts against E. coli revealed active metabolites of the terpene, fatty acid, and polyketide molecular families. However, before our investigations, only the bioactive roimantacene polyene, and p-hydroxyacetophenone amides have been associated with A. sp. While we have demonstrated the potential of observing signal-activated production of functional metabolites in the rhizosphere, our studies provide the condition-specific potential for discovering novel specialized metabolites that would contribute to natural product discovery

Doctor of Philosophy

dissertationOxidative folding is one of the key challenges hampering the development of peptidebased compounds as therapeutics. While disulde-rich peptides are often thought to be more appealing drug lead compounds because of their stable, highly-crosslinked structure, their oxidative folding to the correct disulde connectivity is often dicult, and is optimized in a peptide-specic way. This work advanced knowledge of chemical and biological means to improve oxidative folding of conotoxins. Herein I present a generalized folding protocol suitable for folding diverse disulde-rich peptides. I also show that the incorporation of selenocysteines to replace a disulde bridge with a diselenide eectively adds an intramolecular oxidative folding catalyst, where this bridge had previously been assumed to be static, with any folding improvements being a consequence of conformational eects. These are followed by a discussion of oxidative folding mechanisms in vivo, relating energy expenditure to directing disulde isomerization to the desired connectivity, as well as a novel analysis tool to consider the codon conservation of the cysteine residues that comprise the disulde scaold. This work represents signicant improvements to chemical strategies to eciently produce disulde-rich peptides and genetic analyses towards a better understanding of the role oxidative folding plays in the evolution of disulde scaolds of cysteine-rich peptides

Isolation and characterization of a novel lectin gene, Allium triquetrum agglutin, conferring insecticidal properties against Myzus persicae

Myzus persicae, more commonly known as green peach aphid, is an important pest in agriculture. Each year it causes great losses to agricultural and horticultural plants. It feeds on sap in the phloem, the main transportation route for plant nutrients. It also transmits more than 100 plant viruses, and as there are still no agrichemicals specifically against viruses, farmers often spray large amounts of pesticides to protect their crops. A group of widely-distributed plant proteins, termed lectins, have been recently studied because of their insecticidal properties against aphids. Snowdrop lectin, Galanthus nivalis agglutinin (GNA) was the first isolated monocot mannose-binding lectin, conferring properties of resistance to sap-sucking or homopteran pests. The aim of the project described in this thesis was to identify and express a novel lectin in Arabidopsis thaliana and to assess the performance of the green peach aphid, Myzus persicae, on both transgenic and wild type plants. A gene encoding a novel mannose-binding lectin was cloned from Allium triquetrum (wild garlic). The full-length cDNA of Allium triquetrum agglutinin (ATA) was 719 bp and contained a 522 bp open reading frame encoding a 173 amino acid polypepetide. Homology analysis showed that ATA has high similarity with other mannose-binding lectins and includes three putative mannose-binding subdomains, which suggests that ATA may also confer resistance against aphids. The pGreen 0029 vector and the 35S CaMV cassette were ligated to produce the expression construct. An expression vector was engineered using the pGreen vector together with CaMV 35S promoter and the novel ATA gene was inserted. Transgenic Arabidopsis thaliana plants were subsequently generated using the Agrobacterium-mediated floral dip method. Six homozygous ATA-transformed lines and one empty vector-transformed control line were obtained using kanamycin selection. Several key growth parameters of the transgenic plants were assessed to demonstrate that expressing ATA causes few III phenotypic changes to the host plant. The transgenic plants were subsequently used for aphid bioassays, including choice and non-choice behaviour tests, Mean Relative Growth Rate test, fecundity test, and survival test. The aphid bioassays revealed that (1) ATA does not change the behaviour of aphids to choose a host; (2) Adults are more sensitive to ATA than nymphs; (3) ATA has significant detrimental effects on aphid fecundity. The results described in the thesis show that the production of Allium triquetrum agglutinin in Arabidopsis confers partial resistance to the aphids, Myzus persicae. It is likely that this lectin in related Brassicaceae plants could be used, perhaps in conjunction with other pest-control measures in agriculture. Key words: Allium triquetrum agglutinin, aphid bioassay, Arabidopsis thaliana, choice test, fecundity test, lectin, non-choice test, mean relative growth rate, Myzus persicae, pleiotropic effects, resistance against aphids, survival test

Construction and characterization of live attenuated vaccines in modem lieages of Mycobacterium tuberculosis based on phoP an fadD26 deletions

Tuberculosis es la enfermedad más devastadora producida por un único agente infeccioso. Mycobacterium tuberculosis, el principal agente causal de la tuberculosis en humanos, se transmite entre humanos por el aire. Se estima que en 2017, 1.3 millones de personas (HIV-negativas) murieron a causa de la tuberculosis además de 300, 000 muertes en personas con HIV y se estima que hubo 10 millones de nuevos casos de tuberculosis.A pesar de la existencia de una vacuna preventiva, BCG, con una amplia cobertura de casi el 90% y la disponibilidad de antibióticos para el tratamiento de la tuberculosis, el aumento de cepas resistentes a los antibióticos y la variable eficacia de protección de BCG frente a la tuberculosis pulmonar hace que esta enfermedad sea la primera causa de muerte debido a un agente infeccioso en la actualidad.Para superar este problema, varios nuevos candidatos a vacuna están siendo desarrollados para obtener una vacuna más eficaz. Una de ellas es MTBVAC, una vacuna viva atenuada basada en deleciones en los genes phoP y fadD26 en un aislado clínico perteneciente al linaje 4 de M. tuberculosis. Ambos genes son importantes factores de virulencia de M. tuberculosis.Las micobacterias adaptadas a infectar a humanos presentan una distribución geográfica específica. Son clasificados en siete linajes que pueden diferenciarse entre “modernos” o “ancestrales” basándonos en la deleción TbD1. Los linajes 2, 3 y 4 de M. tuberculosis son los linajes modernos mientras que los linajes 1 y 7 de M. tuberculosis y los linajes 5 y 6 de Mycobacterium africanum son los linajes denominados ancestrales. Los linajes más ampliamente distribuidos son los linajes 2 y 4 de M. tuberculosis seguido de los linajes 1 y 3 de M. tuberculosis con una distribución intermedia y los otros linajes están presentes en determinadas zonas.Teniendo en cuenta que MTBVAC se obtuvo en un aislado clínico del linaje 4 de M. tuberculosis, las mismas deleciones que en MTBVAC en los genes phoP y fadD26 fueron obtenidas en dos aislados clínicos de M. tuberculosis de los linajes 2 y 3, denominadas MTBVAC-L2 y MTBVAC-L3. Para obtener las deleciones se utilizaron dos estrategias diferentes de ingeniería genética. Por lo tanto, estas tres vacunas candidatas basadas en las deleciones de phoP y fadD26 obtenidas en los linajes modernos de M. tuberculosis, permiten la evaluación de la protección dependiente de linaje. Se realizaron análisis de Western-blot y qRT-PCR de los nuevos mutantes dobles para confirmar los fenotipos dependientes de PhoP y FadD26 que han sido descritos previamente en MTBVAC.Se realizó la caracterización preclínica de experimentos de seguridad y eficacia de protección en el modelo de ratón. En experimentos de seguridad, tanto MTBVAC-L2 como MTBVAC-L3 mostraron atenuación, menor que MTBVAC, siendo solo MTBVAC más atenuada que BCG Pasteur en ratones SCID. Para la evaluación de la eficacia en la protección, grupos de ratones fueron vacunados con MTBVAC, MTBVAC-L2, MTBVAC-L3, BCG Pasteur o no vacunados como control. Se evaluó la carga bacteriana en pulmones y bazo en estudios de protección frente a cepas virulentas de los linajes 2, 3 o 4 de M. tuberculosis. MTBVAC, MTBVAC-L2 and MTBVAC-L3 confirieron protección frente a las cepas modernas utilizadas para el desafío sin diferencias significativas entre ellas. Cabe destacar que las cepas MTBVAC y MTBVAC-L2 confirieron una mayor protección frente a la cepa del linaje 2-Beijijng en comparación con BCG Pasteur. Estos resultados sugirieron protección independiente de linaje en ratones y refuerza el conocimiento previo sobre la eficacia de protección de MTBVAC en varios modelos animales.Con objeto de profundizar con la caracterización de MTBVAC, tras estudio de genómica, transcriptómica y proteómica, nos enfocamos en el estudio de metabolitos producidos diferencialmente por MTBVAC. Se observó que la producción del metabolito di-AMP-cíclico (c-di-AMP) era dependiente de PhoPR. El mutante phoPR en H37Rv producía mayor cantidad de c-di-AMP que la cepa silvestre. La secreción del metabolito se observó solo en el mutante phoPR. También se observó una mayor producción y secreción de c-di-AMP en MTBVAC en comparación con la cepa silvestre.El c-di-AMP es un segundo mensajero que está descrito su involucración en diferentes procesos en bacterias. En M. tuberculosis, el c-di-AMP bacteriano es capaz de desencadenar la respuesta de interferón de tipo I (IFN-β) mediante activación del sensor eucariótico STING. Para evaluar si el c-di-AMP de MTBVAC activa esta respuesta en el hospedador, se obtuvieron deleciones en MTBVAC en los genes que codifican para la ciclasa y la fosfodiesterasa del c-di-AMP. Por lo tanto, se obtuvieron tres cepas diferentes de MTBVAC con diferentes niveles de c-di-AMP. A pesar de los aumentados niveles de c-di-AMP bacteriano en MTBVAC, no se observó respuesta de IFN-β tras la infección en células THP-1. En cambio, MTBVAC y los mutantes en la ciclasa y fosfodiesterasa del c-di-AMP mostraron respuesta IL-1β.<br /