CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.Spanish Ministry of Economy and Competitiveness BIO2010-15417 BIO2013-44220-R AGL2013-45339-RRecerCaixa program 2012/ACUP/00048Catalonian government 2017SGR49

“Al inicio creía que el problema era yo”.: Relatos autoetnograficos de profesionales racializados/as en la intervención social.

The fact that racialized people do access the skill labor market, continues to be anecdotical. Very few people have been able to overcome all the bureaucracy trammels and social stigma to obtain a job position. Not to mention to find one within institutions that are public or from the third sector and that acknowledge their vocational training and academic preparation. In this article we analyze some of the aspects that characterize de relationship established between the native professionals and racialized people which occurs in the workspace. The methodological approach implemented is focused on autoethnography and an autobiographical account since we start from the experiences of the authors themselves, in order to seek for visibility and put in debate what has been live but silenced until now. The results show how the social interventions made by racialized professionals are constantly reduced to their origin and their supposed culture, in order to resolve only situations that are not in the range of the native professionals. The background of racialized people is underestimated and instrumentalized during social intervention. Consequently, the workspace becomes an insecure environment, full of suspicious looks, troubling the presence and ignoring the racialized professionals skills and capabilities.El acceso de las personas racializadas al mercado laboral calificado en España continúa siendo un hecho anecdótico. Son pocas las personas que han podido superar las trabas burocráticas y el estigma social para acceder a un puesto de trabajo. Y menos aún, en instituciones públicas o del tercer sector que reconozcan su formación profesional y preparación académica. En este artículo analizamos algunos aspectos que caracterizan la relación que se constituye entre el personal profesional autóctono y el racializado que se da en el espacio laboral. El enfoque metodológico adoptado se centra en el relato biográfico y la auto etnografía puesto que se parte de las experiencias de las propias autoras, buscando de esta manera visibilizar y poner en debate unas experiencias vividas, pero silenciadas hasta el momento. Los resultados muestran cómo las intervenciones sociales realizadas por profesionales racializados/as se ven constantemente reducidas al prisma de su origen y supuesta cultura, para resolver únicamente situaciones que no están al alcance del/la profesional autóctono. El background de los/las profesionales racializados/as se ve menospreciado e instrumentalizando durante la intervención social. Consecuentemente, el espacio laboral, se vuelve un entorno inseguro, lleno de miradas sospechosas, problematizando la presencia e ignorando las capacidades y habilidades de los/las profesionales racializados/as

Differential regulation of CsrC and CsrB by CRP-cAMP in Salmonella enterica

Post-transcriptional regulation mediated by regulatory small RNAs (sRNAs) has risen as a key player in fine-tuning gene expression in response to environmental stimuli. Here, we show that, in Salmonella enterica, the central metabolic regulator CRP-cAMP differentially regulates the sRNAs CsrB and CsrC in a growth phase-dependent manner. While CsrB expression remains unchanged during growth, CsrC displays a growth phase-dependent expression profile, being weakly expressed at the logarithmic growth phase and induced upon entry into stationary phase. We show that CRP-cAMP contributes to the expression pattern of CsrC by repressing its expression during the logarithmic growth phase. The CRP-cAMP mediated repression of CsrC is independent of SirA, a known transcriptional CsrB/CsrC activator. We further show that the sRNA Spot 42, which is derepressed in a Δcrp strain, upregulates CsrC during logarithmic growth. We propose a model where the growth-dependent regulation of CsrC is sustained by the CRP-cAMP-mediated repression of Spot 42. Together, our data point toward a differential regulation of the sRNAs CsrB and CsrC in response to environmental stimuli, leading to fine-tuning of gene expression via the sequestration of the RNA-binding protein CsrA

Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium

The invasion of epithelial cells by Salmonella enterica serovar Typhimurium is a very tightly regulated process. Signaling cascades triggered by different environmental and physiological signals converge to control HilD, an AraC regulator that coordinates the expression of several virulence factors. The expression of hilD is modulated at several steps of the expression process. Here, we report that the invasion of epithelial cells by S. Typhimurium strains lacking the Gre factors, GreA and GreB, is impaired. By interacting with the RNA polymerase secondary channel, the Gre factors prevent backtracking of paused complexes to avoid arrest during transcriptional elongation. Our results indicate that the Gre factors are required for the expression of the bacterial factors needed for epithelial cell invasion by modulating expression of HilD. This regulation does not occur at transcription initiation and depends on the capacity of the Gre factors to prevent backtracking of the RNA polymerase. Remarkably, genetic analyses indicate that the 3'-untranslated region (UTR) of hilD is required for Gre-mediated regulation of hilD expression. Our data provide new insight into the complex regulation of S. Typhimurium virulence and highlight the role of the hilD 3'-UTR as a regulatory motif

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria

Gre factors are required fot biofilm formation in Salmonella enterica serovar Typhimurium by targeting transcription of the cgsD gene

Rdar biofilm formation of Salmonella typhimurium and Escherichia coli is a common ancient multicellular behavior relevant in cell-cell and inter-organism interactions equally, as in interaction with biotic and abiotic surfaces. With the expression of the characteristic extracellular matrix components amyloid curli fimbriae and the exopolysaccharide cellulose, the central hub for the delicate regulation of rdar morphotype expression is the orphan transcriptional regulator CsgD. Gre factors are ubiquitously interacting with RNA polymerase to selectively overcome transcriptional pausing. In this work, we found that GreA/GreB are required for expression of the csgD operon and consequently the rdar morphotype. The ability of the Gre factors to suppress transcriptional pausing and the 147 bp 5′-UTR of csgD are required for the stimulatory effect of the Gre factors on csgD expression. These novel mechanism(s) of regulation for the csgD operon might be relevant under specific stress conditions

Vers una identitat col·lectiva pluricultural (IDCOL)

Aquest document és fruit d'una recerca promoguda l'any 2019 per l'Escola d'Administració Pública de Catalunya en el marc del programa EAPC motor de recerca. Forma part del projecte pilot dut a terme amb la Secretaria d'Igualtat, Migracions i Ciutadania de la Generalitat de Catalunya. Es presenta tal com va ser lliurat pels autors en la seva versió definitiv

CRP-cAMP mediated silencing of virulence expression in Salmonella enterica serovar Typhimurium

[eng] The regulation of the expression of virulence genes in Salmonella enterica serovar Typhimurium is an intensively studied feature of Salmonella lifestyle. How Salmonella integrates environmental signals to activate virulence-related genes within the host has been explored. The genes encoded in Salmonella pathogenicity island I (SPI-1) required for the invasion of epithelial cells are well characterized, and many regulators involved in the activation of SPI-1 genes have been described. However, little is known about mechanism involved in the repression of SPI-1 under conditions were Salmonella does not require the expression of virulence-related genes. The expression of SPI-1 encoded genes have been reported to be a burden for Salmonella physiology and therefore mechanism to control shut down of SPI-1 under non permissive conditions might play a crucial role in Salmonella physiology. Here we describe that at exponential phase, CRP-cAMP, which acts as an activator at stationary phase, represses the expression of SPI-1 genes. The overall objective of this thesis was to characterize how CRP-cAMP silences SPI-1 expression under non-permissive conditions and to describe the molecular mechanism behind this phenotypic observation. In this thesis we i) define the target gene for the CRP-mediated regulation of SPI-1 ii) elucidate at which level of regulation CRP-cAMP modulates the expression of the SPI-1 genes master regulator hilD iii) characterize the involvement of CRP-cAMP dependent sRNA in hilD regulation iv) characterize the interaction of the CRP-cAMP dependent sRNA Spot 42 with the hilD 3’UTR region to regulate SPI-1 and v) explore the role of CRP-cAMP in the modulation of the SPI-1 repressor CsrA through the regulation of the long non-coding RNA csrB and csrC. CRP-cAMP represses hilA expression at exponential phase (non-permissive conditions for SPI-1 expression) and acts as an activator at stationary phase (permissive conditions for SPI-1 expression). CRP-cAMP mediated repression of hilA causes a concomitant attenuation in the expression level of SPI-1 encoded effector proteins. The regulation of SPI-1 during logarithmic growth phase occurs upstream of HilA by repressing hilD, hilC and rtsA expression and is mediated by the regulation of hilD expression at the post transcriptional level through the hilD 3’UTR. CRP-cAMP mediated regulation of hilD requires, in addition to the hilD 3’UTR, the sRNA chaperone Hfq and the major endonuclease RNAse E. CRP-cAMP represses the expression of the sRNA Spot 42 at exponential phase. We show that Spot 42 positively regulates hilD expression at exponential growth phase and requires of the presence of the hilD 3’UTR, the sRNA chaperone Hfq and the major endonuclease RNAse E. Interestingly, Spot 42 and the hilD 3’UTR region physically bind to Hfq. Spot 42 physically interacts with the last 150 nt of the hilD 3’UTR and unstructured region III of Spot 42 is required for the regulation of hilD. CRP-cAMP represses csrC but not csrB expression at exponential phase to regulate the expression of hilD. Remarkably, the CRP-cAMP dependent sRNA Spot 42 positively regulates the expression of csrC.[spa] La regulación de la expresión de genes de virulencia en Salmonella enterica serovar Typhimurium es una característica intensamente estudiada en Salmonella. La forma en que Salmonella integra señales ambientales para activar los genes relacionados con la virulencia dentro del huésped ha sido explorada. Los genes codificados en la isla de patogenicidad I de Salmonella (SPI-1) son necesarios para la invasión de células epiteliales, están bien caracterizados y se han descrito muchos reguladores implicados en su activación. Sin embargo, poco se sabe sobre el mecanismo implicado en la represión de SPI-1 en condiciones en las que Salmonella no requiere la expresión de genes relacionados con la virulencia. Es sabido que la expresión de genes codificados por SPI-1 son una carga para la fisiología de Salmonella y por lo tanto el mecanismo para controlar la represión de SPI-1 en condiciones no permisivas podría desempeñar un papel crucial en la fisiología de Salmonella. Aquí se describe que en fase exponencial, CRP-cAMP, que actúa como un activador en fase estacionaria, reprime la expresión de los genes SPI-1. El objetivo general de esta tesis fue caracterizar cómo el CRP-cAMP silencia la expresión de SPI-1 en condiciones no permisivas y describir el mecanismo molecular detrás de esta observación fenotípica. El CRP-cAMP reprime la expresión de hilA en la fase exponencial (condiciones no permisivas para la expresión de SPI-1) y actúa como un activador en fase estacionaria (condiciones permisivas para la expresión de SPI-1). La represión mediada por CRP-cAMP de hilA provoca una atenuación concomitante en el nivel de expresión de proteínas efectoras codificadas por SPI-1. La regulación de SPI-1 durante la fase de crecimiento logarítmico se produce aguas arriba de HilA mediante la represión hilD, hilC y rtsA expresión y está mediada por la regulación de hilD a nivel post transcripcional a través de la hilD 3'UTR. La regulación mediada por CRP-cAMP de hilD requiere, además de la hilD 3'UTR, la chaperona Hfq y la endonucleasa RNAsa E. CRP-cAMP reprime la expresión del sRNA Spot 42 en la fase exponencial. Mostramos que Spot 42 regula positivamente la expresión hilD en la fase de crecimiento exponencial, Spot 42 interacciona físicamente con los últimos 150 nt de la hilD 3'UTR

Mares d'origen marroquí a Catalunya : entre les experiències personals i els processos d'acompanyament a l'escolarització dels fills i filles

La present tesi doctoral planteja un objectiu doble, per una banda, conèixer les experiències de les mares d'origen marroquí a Catalunya, tot identificant les dimensions que poden explicar la seva relació amb l'escola i la comunitat educativa i, en segon lloc, analitzar les formes i estratègies d'acompanyament que realitzen durant l'etapa d'escolarització obligatòria dels seus fills i filles. La recerca s'inscriu en la línia dels treballs que s'han interessat en estudiar la relació família-escola però des d'una vessant que situa a les famílies, i en concret a les mares com a focus central. El treball supera les formes en que tradicionalment s'entén des dels centres escolars la participació i la implicació familiar, opta per visibilitzar el paper de les mares en la vida escolar dels fills i filles i busca destacar les estratègies diàries poc reconegudes vers l'acompanyament que les mares impulsen. La recerca adopta una perspectiva interseccional i multidimensional i articula variables diverses; posa de relleu aquelles que resulten pròpies i intrínseques al propi col·lectiu, com són el gènere, la classe social, el país d'origen i la maternitat, mentre que d'altres són resultat de les conjuntures geopolítiques i les dimensions estructurals, on prenen rellevància les relacions dominants Nord - Sud. La imbricació de tots aquests elements es posen en joc a l'espai escolar. L'objectiu de la tesi ha determinat la metodologia qualitativa i el relat biogràfic com l'apropament més apropiat. Aquest, ha estat complementat amb observació participant presencial i a través de les xarxes socials, la netnografia. L'enfocament interpretatiu proposat ha permès analitzar el fenomen des de la perspectiva de les protagonistes. La investigadora ha estat privilegiada al poder incorporar a aquesta posició la d'immigrant d'origen marroquí, amb fills en edat escolar, treballadora d'una institució pública i la seva experiència com a formadora en centres escolars. Els resultats obtinguts mostren la singularitat dels itineraris migratoris i la heterogeneïtat dels perfils de les mares, així com l'existència de barreres i dificultats que caracteritzen en bona mesura la seva relació amb el sistema escolar, situació que es manifesta en diferents moments de la trajectòria escolar dels seus filles i fills. La tesi presenta com les mares materialitzen de formes diverses el sentit originari del terme estar al costat i compartir el camí, però com en tots els casos, l'acompanyament constitueix l'eix principal de les seves quotidianitats, transcendeix la seva presència física i el suport acadèmic, i assoleix un sentit relacional, emocional i preventiu davant un entorn que perceben hostil. Hem pogut construir tipologies diferenciades vers aquest acompanyament, el que s'enfoca de forma exclusiva sobre l'infant o el que resulta un acompanyament holístic que, a més d'enfocar-se sobre l'infant de forma directe, també pretén actuar sobre l'entorn. Les mares que aposten per aquest acompanyament estan presents en els diferents òrgans de l'escola i/o estan presents també en espais de militància social contra la discriminació i la injustícia social.Esta tesis doctoral establece un doble objetivo, por un lado, conocer las experiencias de las madres de origen marroquí en Cataluña, identificando las dimensiones que pueden explicar su relación con la escuela y la comunidad educativa y, en segundo lugar, analizar las formas y estrategias de acompañamiento que llevan a cabo durante la etapa de escolarización obligatoria de sus hijos. La investigación se inscribe en la línea de trabajos que se han interesado en estudiar la relación familia-escuela, pero desde una perspectiva que coloca a las familias, y específicamente a las madres , como el foco central. El trabajo supera las formas en que la participación y la participación de la familia se entiende tradicionalmente desde las escuelas, elige hacer visible el papel de las madres en la vida escolar de los hijos e hijas y busca destacar las estrategias diarias poco reconocidas hacia el acompañamiento que las madres promueven. La investigación adopta una perspectiva interseccional y multidimensional y articula diversas variables; destaca aquellas que son propias e intrínsecas al propio colectivo, como el género, la clase social, el país de origen y la maternidad, mientras que otras son el resultado de coyunturas geopolíticas y dimensiones estructurales, donde las relaciones dominantes Norte -Sur toman relevancia. La imbricación de todos estos elementos se pone en juego en el ámbito escolar. El objetivo de la tesis ha determinado la metodología cualitativa y la historia biográfica como el enfoque más adecuado. Esto se ha complementado con la observación participando en persona y a través de redes sociales, netnografía. El enfoque interpretativo propuesto ha permitido analizar el fenómeno desde la perspectiva de las protagonistas. La investigadora ha tenido el privilegio de poder incorporar a esta posición la de inmigrante de origen marroquí, con hijos en edad escolar, trabajadora de una institución pública y su experiencia como formadora en centros escolares. Los resultados obtenidos muestran la singularidad de los itinerarios migratorios y la heterogeneidad de los perfiles de las madres, así como la existencia de barreras y dificultades que caracterizan en gran medida su relación con el sistema escolar, situación que se manifiesta en diferentes momentos de la trayectoria escolar de sus hijas e hijos. La tesis presenta cómo las madres se materializan de diferentes maneras el sentido original de la expresión al lado y compartiendo el camino, pero como en todos los casos, el acompañamiento constituye el eje principal de su vida cotidiana, trasciende su presencia física y apoyo académico, y logra un sentido relacional, emocional y preventivo en un entorno que perciben hostil. Hemos sido capaces de construir tipologías diferenciadas hacia este acompañamiento, que se centra exclusivamente en el niño/a o lo que es un acompañamiento holístico que, además de centrarse directamente en el niño/a, también pretende actuar sobre el entorno. Las madres comprometidas con este acompañamiento están presentes en los diferentes órganos de la escuela y/o también están presentes en espacios de militancia social contra la discriminación y la injusticia social.This doctoral thesis has a dual aim. On the one hand, to get to know the experiences of mothers of Moroccan origin within Catalonia, identifying the elements that can explain their relationship with the school and with the educational community. On the other, to analyse their methods and strategies for accompanying their children during their period of compulsory education. The research forms part of the line of investigations that have studied the family-school relationship, but with a perspective that places families, and in particular mothers, as a central focus. This thesis goes beyond the ways in which family participation and involvement have traditionally been understood by schools, choosing to make visible the role of mothers in their children's schooling and seeking to highlight the often unrecognised day to day strategies that mothers use in accompanying their children. The study adopts an intersectional and multidimensional perspective, relating different variables. It highlights those that are specific and intrinsic to the group itself, such as gender, social class, country of origin and motherhood, while others are the product of geopolitical circumstances and structural dimensions: here the relations of dominance between the global North and South show their importance. All these elements interweave and come into play within the educational environment. The aim of the thesis has dictated a qualitative methodology and the biographical account as the most appropriate approach. This has been complemented with face-to-face participant observation and the analysis of social media, netnography. The interpretive approach adopted has made it possible to analyse the phenomenon from the perspective of the protagonists. The researcher has been privileged to be able to incorporate into this her position as a migrant of Moroccan origin, with children of school age, working in a public institution and with experience as an educator in schools. The results obtained show the uniqueness of migratory itineraries and the heterogeneity of mothers' profiles, as well as the existence of barriers and difficulties that strongly influence their relationship with the school system, a situation that manifests itself at different times throughout their children's schooling. The thesis shows how mothers materialise in diverse ways the original meaning of the term to be by their side and share their path. However, as in all cases this accompaniment is the central axis of their daily lives, it goes beyond their physical presence and academic support, and takes on a relational, emotional, and preventive aspect in the face of an environment they perceive to be hostile. We have been able to establish different typologies in this accompaniment: those which focus exclusively on the child itself; and others that involve a more holistic accompaniment which, in addition to focusing directly on the child, also aim to act on the environment. Those mothers who opt for this accompaniment strategy participate in the different organs of the school and/or in spaces of social activism against discrimination and social injustice.Universitat Autònoma de Barcelona. Programa de Doctorat en Educaci

3' untranslated regions: regulation at the end of the road

Post-transcriptional gene regulation in bacteria plays a major role in the adaptation of bacterial cells to the changing conditions encountered in the environment. In bacteria, most of the regulation at the level of mRNA seems to be targeting the 5′untranslated regions where accessibility to the ribosome-binding site can be modulated to alter gene expression. In recent years, the role of 3′untranslated regions has gained attention also as a site for post-transcriptional regulation. In addition to be a source of trans-encoded small RNAs, the 3′untranslated regions can be targets to modulate gene expression. Taking recent findings in the post-transcriptional regulation of the hilD gene, encoding for the main regulator of virulence in Salmonella enterica serovar Typhimurium, we highlight the role of 3′untranslated regions as targets of post-transcriptional regulation mediated by small RNAs and discuss the implications of transcriptional elongation in the 3′UTR-mediated regulation in bacteria