Tesis (Microbiología)https://hdl.handle.net/11441/109072024-03-28T11:49:24Z2024-03-28T11:49:24ZCaracterización de los genes implicados en la síntesis de ceras en semillas de girasol (Helianthus annuus)https://hdl.handle.net/11441/1563582024-03-18T10:35:14Z2024-01-19T00:00:00ZCaracterización de los genes implicados en la síntesis de ceras en semillas de girasol (Helianthus annuus)
El girasol (Helianthus annuus L.) es una planta oleaginosa de alto valor comercial. El principal producto
derivado de su cultivo es el aceite extraído de sus semillas, destinado principalmente al consumo
humano. El aceite de girasol debe someterse a un proceso de refinado para eliminar compuestos
minoritarios y adecuar el producto final a las exigencias del consumidor. Las ceras son un subproducto
del proceso de refinación, y sus propiedades fisicoquímicas las convierten en compuestos de gran
interés industrial.
Las ceras son componentes de las capas lipídicas de la cutícula de las plantas, y desempeñan un papel
crucial en su adaptación y supervivencia. En el girasol, las ceras se encuentran principalmente en el
pericarpio o cubierta de la semilla, donde contribuyen a la protección del embrión y la viabilidad de la
semilla. La síntesis de ceras en las plantas tiene lugar en el retículo endoplasmático a partir de
precursores de ácidos grasos de cadena muy larga (VLCFA), e implica dos reacciones enzimáticas
consecutivas. En primer lugar, los VLCFA se reducen a alcoholes grasos de cadena larga por las
reductasas de ácidos grasos (FAR). A continuación, estos alcoholes de cadena larga se esterifican con
moléculas de acil-CoA por las sintasas de ceras (WS), para formar ceras como producto final. Los
avances en la identificación y caracterización de los genes responsables de la síntesis de ceras en
diversos organismos ofrecen la posibilidad de utilizar procesos biotecnológicos para lograr una
producción sostenible y a gran escala de estos compuestos en bacterias, levaduras o plantas
oleaginosas.
El objetivo general de este trabajo es la caracterización de las enzimas implicadas en la biosíntesis de
ceras en semillas de girasol, las reductasas de ácidos grasos y sintasas de ceras, así como el desarrollo
de un nuevo método preciso de análisis de ceras de girasol. Se han identificado y estudiado 4 genes
FAR (HaFAR2, HaFAR3, HaFAR4 y HaFAR5) y 2 genes WS (HaWS8 y HaWS11), aquellos que
mostraron mayores niveles de expresión en semillas de girasol, y dichos genes se han expresado
heterólogamente en Saccharomyces cerevisiae para comprobar su funcionalidad y evaluar su impacto
en el perfil lipídico. Además, se han generado plantas transgénicas de Arabidopsis thaliana que
sobreexpresaban HaFAR2, y se ha analizado la composición lipídica de sus hojas. También se ha
estudiado la actividad de las enzimas HaWS in vitro, y determinado sus constantes cinéticas. Asimismo,
se han modelado in silico las estructuras de cada proteína con sustratos específicos, y se ha estudiado
su localización subcelular mediante expresión transitoria en hojas de Nicotiana benthamiana. Por último,
se ha estudiado el perfil de acumulación de ceras en las cubiertas de semillas de girasol, mostrando un
incremento en su acumulación a lo largo del desarrollo de la semilla. Además. se ha desarrollado un
nuevo método de determinación de ceras basado en la extracción directa de ceras de la superficie de las
semillas de girasol y su análisis mediante cromatografía de gases acoplado a masas, aplicable también
a la determinación de ceras a partir de aceite crudo de girasol. El papel de estas enzimas en la síntesis
de las ceras presentes en la semilla de girasol se evaluó en base a los resultados obtenidos.
2024-01-19T00:00:00ZEscherichia coli infection: mechanisms and rapid detection of resistance to the association of ß-lactams with ß-lactamase inhibitors, and impact of the Sars-Cov-2 pandemic on the morbidity and mortality of intra-abdominal infectionhttps://hdl.handle.net/11441/1556852024-02-29T09:39:13Z2023-11-03T00:00:00ZEscherichia coli infection: mechanisms and rapid detection of resistance to the association of ß-lactams with ß-lactamase inhibitors, and impact of the Sars-Cov-2 pandemic on the morbidity and mortality of intra-abdominal infection
Infections by Escherichia coli in human beings produce a significant disease burden, with important morbidity and mortality, and the increasing resistance to antimicrobials is a challenge for choosing the more effective therapy. In this context, the present PhD Thesis is aimed to produce new knowledges applicable to the treatment of one of the more frequent and severe infections by E. coli¸ the intra-abdominal infections.
Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among E. coli isolates. The first objective was to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analysed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). A higher copy number of blaTEM gene in P/T-R isolates was found, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC.
Currently, the detection of P/T-R relying on conventional methods is time-consuming. To overcome this issue, a cost-effective test based on MALDI-MS technology has been developed, which aims to detect P/T-R and extended-spectrum resistance to ß-lactam/ß-lactamase inhibitors (ESRI) in E. coli. Automated Clover MS Data Analysis software to analyse the protein profile spectra obtained by MALDI-MS from a collection of 248 E. coli isolates (91 P/T-resistant, 81 ESRI developers and 76 P/T-susceptible) has been used. This software allowed to preprocess all the spectra to build different peak matrices that were analysed by machine learning algorithms. The test can efficiently and rapidly (15 min) discriminate between P/T-resistant, ESRI developer and P/T-susceptible isolates and allowed the correct classification between ESRI developers from their isogenic resistance to P/T.
Finally, the COVID-19 pandemic by the new SARS-CoV-2 arose new questions derived from the great challenge to the Health Systems and, specifically, in the case of the intra-abdominal infections, about of the impact of the pandemic on the management of them, and looking for data to also improve the clinical approach in these cases.
The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms. Overall, the data provided by this PhD thesis point to the importance of understanding the mechanisms of resistance acquisition and its early detection in order to have an impact on mortality and morbidity in severe infections.
2023-11-03T00:00:00ZEstudio de la ubicuitilación del complejo MRN mediada por SCF(bTrCP/FBXW7): implicaciones biológicashttps://hdl.handle.net/11441/1491932023-09-28T09:05:13Z2023-07-07T00:00:00ZEstudio de la ubicuitilación del complejo MRN mediada por SCF(bTrCP/FBXW7): implicaciones biológicas
El cisplatino, como otros fármacos basados en platino, se encuentra entre los
compuestos más usados en las quimioterapias contra distintos tipos de cánceres. Entre
sus múltiples efectos, el cisplatino se une al ADN generando una gran variedad de
lesiones que provocan, en última instancia, la muerte celular. Algunas de estas
lesiones, en concreto las roturas de doble cadena, pueden ser reparadas por
recombinación homóloga o por unión de extremos no homólogos, provocando
resistencias al fármaco. Uno de los principales intervinientes en la reparación de las
roturas de doble cadena es el complejo MRN (MRE11, RAD50 y NBS1), esencial para la
identificación de las roturas, el reclutamiento de diversos factores de señalización y el
procesamiento, todo ello encaminado a la reparación de las mismas. El complejo MRN
también interviene en otros procesos, como la reparación del ADN tras la formación
de aductos o tras el colapso de horquillas de replicación. Dado su papel en el
mantenimiento de la estabilidad del genoma, los defectos en los componentes del
complejo MRN se han asociado con enfermedades como el cáncer.
La ubicuitilación de proteínas es una modificación post-traduccional que
desempeña importantes funciones en la célula: desde la degradación de proteínas vía
proteasoma o lisosoma, hasta la modificación de la localización de proteínas concretas
o participando en procesos de señalización. La ubicuitilación de las proteínas tiene un
papel esencial en el mantenimiento de la homeostasis celular. Por ello, las alteraciones
en la ubicuitilación de determinadas proteínas pueden contribuir a la transformación
tumoral. En el proceso de ubicuitilación intervienen distintas enzimas, entre las que
destaca la ligasa de ubicuitina, por ser la encargada del reconocimiento del sustrato. La
ligasa de ubicuitina SCF está formada por una serie de subunidades entre las que se
encuentran las proteínas F-box, cuyo papel es identificar a los sustratos específicos de
esta ligasa. Dos de las proteínas F-box más destacadas de SCF son TrCP y FBXW7.
Ambas son responsables de la ubicuitilación de una gran variedad de sustratos
implicados en múltiples procesos celulares, y tanto estos como dichas proteínas F-box
se han relacionado con diferentes aspectos de la transformación tumoral.
En esta Tesis nos hemos centrado en el estudio del complejo MRN como potencial
sustrato de SCF(TrCP/FBXW7). En el laboratorio se había realizado un estudio de las
proteínas que interaccionaban con TrCP y FBXW7 mediante espectrometría de
masas, y entre ellas estaban algunos de los componentes del complejo MRN. Ahora
hemos podido confirmar que existe una asociación in vivo entre estas proteínas F-box
y el complejo MRN en el núcleo de las células de mamífero. Además, tanto SCF(TrCP)
como SCF(FBXW7) son capaces de ubicuitilar in vitro e in vivo ciertos componentes del
complejo, por lo que este es sustratos de ambas ligasas de ubicuitina. La asociación
entre TrCP y MRN ocurre a través de MRE11, que es la diana in vivo de esta proteína
F-box, y está regulada por las quinasas p70S6K y GSK3. Esta última quinasa también
interviene en la asociación de FBXW7 con el complejo MRN. Profundizando en el papel fisiológico de las asociaciones entre estas proteínas Fbox
y el complejo MRN, pusimos de manifiesto que TrCP estimula la localización de
los componentes del complejo en la cromatina, aunque se requieren más estudios
para conocer la relevancia de estos resultados. Por su parte, la interacción entre
FBXW7 y el complejo MRN induce la degradación del mismo por la vía de la
autofagia/lisosoma. La ubicuitilación llevada a cabo por SCF(FBXW7) permite que el
complejo MRN se asocie con los mediadores del flujo autofágico, p62 y LC3, en el
núcleo de la célula para, posteriormente, trasladarse a los lisosomas, donde se llevará
a cabo su degradación. Este proceso se potencia cuando la célula entra en apoptosis
derivada del tratamiento con agentes genotóxicos.
Por último, a raíz de los ensayos que realizamos estudiando el comportamiento del
complejo MRN en respuesta a cisplatino, observamos en varias líneas celulares que el
tratamiento con dosis subletales de cisplatino provocaba su entrada en senescencia.
Sin embargo, en las líneas celulares equivalentes que sobreexpresaban TrCP se
producía muerte celular, mostrando además unos niveles de p21 CIP1 inferiores a los
de las correspondientes líneas silvestres. En la Tesis discutimos las implicaciones
derivadas de todos estos resultados.
2023-07-07T00:00:00ZOptimisation of the therapeutic potential of fosfomycin against Enterobacteriaceae: characterisation of genetic and physiological factors related to resistance and antimicrobial activityhttps://hdl.handle.net/11441/1481402024-02-13T21:59:26Z2023-05-30T00:00:00ZOptimisation of the therapeutic potential of fosfomycin against Enterobacteriaceae: characterisation of genetic and physiological factors related to resistance and antimicrobial activity
Since the discovery of the first antimicrobials, bacteria with resistance mechanisms
against them have been detected. The appearance of bacterial resistance is a natural
phenomenon, which has increased as a result of the use of antimicrobials. Therefore, the
availability of antimicrobials does not ensure therapeutic success. Moreover, in recent decades,
a progressive increase in antimicrobial resistance has occurred, and it has become a global public
health problem, since there is an increase in deaths caused by or related to bacteria that present
resistance mechanisms. As a result of this problem and the scarcity of new effective molecules
for the treatment of multidrug-resistant bacteria, various organizations (such as WHO and FAO)
are developing plans with different strategies to address the problem. These strategies include
optimizing of the use of existing antimicrobials and the rescue of old antibiotics that are still
active, such as fosfomycin.
Fosfomycin is an old antimicrobial that can be a good therapeutic option, since it many
bacteria of clinical interest remain sensitive to this antibiotic. Fosfomycin is a derivative of
phosphonic acid, a hydrophilic, low molecular weight molecule. It has three carbon atoms, is
soluble in water, and is similar to phosphoenolpyruvate. It is a broad-spectrum bactericidal
antimicrobial that acts in the growth phase of bacteria, inhibiting the first step of cell wall
peptidoglycan synthesis by binding to the enzyme MurA. Fosfomycin must penetrate the
cytoplasm to reach its target, MurA, producing bactericidal effect. For this purpose, two
membrane transporters GlpT and UhpT are described, whose physiological function in bacteria is
the uptake of phosphorylated carbon sources and expel inorganic phosphate (Pi). The regulation
and activity of these transporters is fundamental to the mechanism of action of fosfomycin and,
therefore, to fosfomycin resistance.
The transcription of both transporters is induced by their own substrate, in addition to
the AMPc-CRP metabolism regulator complex, and they are also activated by the FNR regulator,
a bacterial regulator under anaerobic conditions. The GlpT transporter has the function of
introducing glycerol-3-phosphate (G3P), this molecule binds to the GlpR repressor, causing the
loss of affinity for promoters of the glp regulon genes, such as glpT. On the other hand, UhpT
transports hexose-phosphate, mainly glucose-6-phosphate (G6P). This molecule is detected by a
two-component system, UhpB and C, and when this occurs, it phosphorylates UhpA, which binds
to the uhpT promoter, inducing its transcription. Thus, in the susceptibility assays G6P must be
added to induce the presence of this transporter, as the susceptibility results obtained in this way
are more consistent with susceptibility breakpoints and therapeutic success.
Fosfomycin resistance mechanisms can be plasmid and chromosomal mediated, as
modifications of MurA, the presence of peptidoglycan recycling pathways, alteration of
fosfomycin permeability or the presence of fosfomycin-modifying enzymes. Chromosomal
mediated fosfomycin resistance usually occurs in a stepwise mode, often generating complex
phenotypes difficult to interpret.
In this sense, to better understand the mechanisms of resistance to fosfomycin in
Klebsiella pneumoniae and to optimize the use of this antimicrobial, the following study was
carried out. The objectives were to characterize the role of the genes uhpT, glpT, and fosA in
resistance to fosfomycin in K. pneumoniae and to evaluate the use of phosphonoformate sodium
(PPF) due to its ability to inhibit the FosA enzyme, in combination with fosfomycin. For this
purpose, seven clinical isolates of K. pneumoniae and the reference strain (ATCC 700721) were
used, and their genomes were sequenced. Mutants for transporters and fosA were constructed
from two isolates of K. pneumoniae ATCC 700721. The susceptibility test to fosfomycin was
performed using the gradient strip method. Synergy between fosfomycin and PPF was studied by
checkerboard assay and analyzed with SynergyFinder. Spontaneous frequencies of occurrence of
fosfomycin and PPF mutants, in vitro activity by growth curves with gradient concentrations of
fosfomycin with and without PPF, and time-kill assays with and without PPF were also evaluated.
The fosfomycin MICs of the clinical isolates ranged from 16 to 1,024mg/L. The addition of 0.623
mM PPF reduced the MIC by 2 to 8-times. Deletion of fosA gene led to a 32-fold decrease.
Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic
area at 0.623mM). The lowest frequencies of fosfomycin resistant mutants were found in ΔfosA
mutants with frequency ranging from 1.69x10-1 to 1.60x10-5 for 64 mg/L fosfomycin. Finally, the
growth monitoring and time-kill assays, fosfomycin showed bactericidal activity only against fosA
mutants and not with the addition of PPF. The study concludes that inactivation of the fosA gene
results in decreased resistance to fosfomycin in K. pneumoniae. The pharmacological approach
using PPF did not achieve sufficient activity and the effect decreased with the presence of other
fosfomycin resistant mutations.
The second chapter of the Thesis follows the line of optimizing the use of fosfomycin with
the addition of an adjuvant, and to better understand how the regulation of fosfomycin
transporters may affect their activity. The main objective was to evaluate the role of glycerol at
therapeutically relevant concentrations in combination with fosfomycin in Escherichia coli, since
this molecule is clinically used as a treatment for example for elevated intracranial pressure and
can induce glpT expression. For this purpose, a collection of isogenic mutants of fosfomycinrelated genes was evaluated in E. coli strains. The induction of fosfomycin transporters was
evaluated and susceptibility tests, interaction assays, and time-to-death assays were performed.
Our results showed that glycerol allows the activation of the GlpT transporter, but this induction
is delayed in time and is not homogeneous in all E. coli strains throughout the bacterial
population, leading to contradictory results in terms of fosfomycin activity. The susceptibility
assays showed increased fosfomycin activity with glycerol in the disc diffusion assay, but not in
the agar dilution or broth microdilution assays. Similarly, in time-kill assays, the effect of glycerol
was absent because of the appearance of fosfomycin-resistant subpopulations. In conclusion,
glycerol may not be a good candidate for use as an adjuvant to fosfomycin.
Finally, to better understand physiological factors that affect fosfomycin transporters
activity, the aim of third chapter was to evaluate the in vitro activity of fosfomycin under different
physiological concentrations of inorganic phosphate (Pi). For this purpose, the wild-type strain
BW25113, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT and ΔphoB) and six clinical isolates
of E. coli with different fosfomycin susceptibilities were used. Susceptibility was assessed by agar
dilution using Mueller-Hinton agar (Pi=1mM) and supplemented with Pi (13 and 42mM, minimum
and maximum urinary concentrations of Pi) and/or glucose-6-phosphate (25mg/L). The promoter
activity of the fosfomycin transporter was assessed by monitoring fluorescence accumulation
using pUA66-PglpT::gfpmut2 or pUA66-PuhpT::gfpmut2 plasmids in standard Mueller-Hinton
broth (MHB) supplemented with Pi (13 or 42mM) ± glucose-6-phosphate. Fosfomycin activity was
quantified spectrophotometrically at 24 hours as before with glucose-6-phosphate, and
fosfomycin ranged from 1 to 1024mg/L. The EC50 of fosfomycin was estimated and compared.
Time-kill assays were performed with fosfomycin concentrations of 307 (plasma Cmax), 1053 and
4415mg/L (urinary Cmax range), using MHB with 28mM Pi (mean urinary concentration) +25mg/L
glucose-6-phosphate. The results showed that all strains decreased fosfomycin susceptibility
linked to increasing Pi concentrations: 1-4-log2 dilution differences from 1 to 13mM, and 1-8-log2
dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the
expression of fosfomycin transporter promoters. Also, increasing Pi concentrations resulted in a
higher bacterial viability EC50 of fosfomycin, except against the ΔglpT-uhpT mutant strain.
Therefore, the present study concludes that Pi variations in physiological fluids may reduce the
activity of fosfomycin against E. coli. Also, the elevated urinary Pi concentrations may explain the
failure of oral fosfomycin in non-wild but fosfomycin-susceptible E. coli strains.
2023-05-30T00:00:00Z