Contribution of phenotypic heterogeneity to adaptive antibiotic resistance

Antibiotic-resistant isolates of Salmonella enterica were selected on plates containing lethal concentrations of rifampicin, kanamycin, and nalidixic acid. The stability of the resistance phenotype was scored after nonselective growth. Rifampicin-resistant (Rifr) isolates were stable, suggesting that they had arisen by mutation. Mutations in the rpoB gene were detected indeed in Rifr mutants. In contrast, a fraction of kanamycin-resistant (Kmr) and nalidixic acid-resistant (Nal r) isolates showed reduced resistance after nonselective growth, suggesting that mechanisms other than mutation had contributed to bacterial survival upon lethal selection. Single-cell analysis revealed heterogeneity in expression of the porin gene ompC, and subpopulation separation provided evidence that reduced ompC expression confers adaptive resistance to kanamycin. In the case of Nalr isolates, mutations in the gyrA gene were present in most nalidixic acid-resistant isolates. However, the efflux pump inhibitor Phe-Arg-β-naphtylamide (PAβN) reduced the level of resistance in Nalr mutants, indicating that active efflux contributes to the overall level of nalidixic acid resistance. Heterogeneous efflux pump activity was detected in single cells and colonies, and a correlation between high efflux and increased resistance to nalidixic acid was found. These observations suggest that fluctuations in the expression and the activity of critical functions of the bacterial cell, alone or combined with mutations, can contribute to adaptive resistance to antibiotics.Ministerio de Economía e Innovación BIO2010-15023, CSD 2008-00013Junta de Andalucía. Consejería de Innovación, Ciencia y Empresa. CVI-587

Contribution of SPI-1 bistability to Salmonella enterica cooperative virulence: insights from single cell analysis

Salmonella enterica pathogenicity island 1 (SPI-1) is a gene cluster that encodes a type III secretion system and effectors involved in epithelial cell invasion. SPI-1 undergoes bistable expression, with concomitant formation of SPI-1ON and SPI-1OFF lineages. This study describes single cell analysis of SP1-1 bistability and epithelial cell invasion, and reports the unsuspected observation that optimal invasion of epithelial cells requires the presence of both SPI-1ON and SPI-1OFF subpopulations. The contribution of SPI-1OFF cells to optimal invasion may rely on their ability to invade epithelial cells if a SPI-1ON subpopulation is present. In fact, Salmonella SPI-1 mutants are also able to invade epithelial cells in the presence of SPI-1ON Salmonellae, a phenomenon described in the 1990’s by Galán and co-workers. Invasion by SPI-1OFF cells does not seem to involve a diffusible factor. A small number of SPI-1ON cells is sufficient to endow the bacterial population with invasion capacity, a feature that may permit host colonization regardless of the bottlenecks encountered by Salmonella populations inside animals.España, MINECO BIO2016-75235-

Waddington's Landscapes in the Bacterial World

Conrad Waddington's epigenetic landscape, a visual metaphor for the development of multicellular organisms, is appropriate to depict the formation of phenotypic variants of bacterial cells. Examples of bacterial differentiation that result in morphological change have been known for decades. In addition, bacterial populations contain phenotypic cell variants that lack morphological change, and the advent of fluorescent protein technology and single-cell analysis has unveiled scores of examples. Cell-specific gene expression patterns can have a random origin or arise as a programmed event. When phenotypic cell-to-cell differences are heritable, bacterial lineages are formed. The mechanisms that transmit epigenetic states to daughter cells can have strikingly different levels of complexity, from the propagation of simple feedback loops to the formation of complex DNA methylation patterns. Game theory predicts that phenotypic heterogeneity can facilitate bacterial adaptation to hostile or unpredictable environments, serving either as a division of labor or as a bet hedging that anticipates future challenges. Experimental observation confirms the existence of both types of strategies in the bacterial world.España Ministerio de Ciencia e Innovación Grant BIO2016- 75235-

Single cell analysis of bistable expression of pathogenicity island 1 and the flagellar regulon in Salmonella enterica

Bistable expression of the Salmonella enterica pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1OFF FlagOFF, SPI-1OFF FlagON, SPI-1ON FlagOFF, and SPI-1ON FlagON. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that FlagOFF cells might contribute to optimal invasion as previously proposed for SPI-1OFF cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1ON cells. As a consequence, growth resumption in SPI-1ON FlagON cells requires switching to both SPI-1OFF and FlagOFF states.Ministerio de Economía, Industria y Competitividad. Gobierno de España-BIO2016-75235-PFondo Europeo de Desarrollo Regional. Unión EuropeaVI Plan Propio de Investigación y Transferencia. Universidad de Sevilla. Españ

Learning how our Body Responds to Infections: a New Approach to Teaching Immunology

Este trabajo presenta una experiencia docente en el aula impartida en la asignatura de Biología de primer curso del Grado en Farmacia. El ciclo de mejora en el aula desarrollado propone un cambio del modelo teórico clásico basado en clases magistrales hacia un modelo fundamentado en el aprendizaje crítico natural. El tema abordado corresponde a conceptos básicos de inmunología. El objetivo principal ha sido dar protagonismo a los alumnos durante el proceso de aprendizaje, mediante una presencia activa del profesor al mismo tiempo que discreta. Los cuestionarios han sido una herramienta fundamental en el proceso para conocer la evolución de los modelos mentales de los estudiantes y los resultados fueron muy satisfactorios, proporcionando al alumnado una experiencia de aprendizaje muy enriquecedora.This work presents a teaching experience in the classroom taught in the biology subject of the first year of the Degree in Pharmacy. The improvement cycle proposes a change from the classical theoretical model based on lectures to a model based on natural critical learning. The topic addressed corresponds to basic concepts of immunology. The main objective has been to give prominence to the students during the learning process, through an active presence of the teacher at the same time as discreet. The questionnaires have been a fundamental tool in the process to know the evolution of the mental models of the students and the results were very satisfactory, as well as a very enriching learning experience for the students

Regulation of bistability in the std fimbrial operon of Salmonella enterica by DNA adenine methylation and transcription factors HdfR, StdE and StdF

Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.Ministerio de Ciencia, Innovación y Universidades [BIO2016–75235-P

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

Distinct Microglial Responses in Two Transgenic Murine Models of TAU Pathology

Microglial cells are crucial players in the pathological process of neurodegenerative diseases, such as Alzheimer’s disease (AD). Microglial response in AD has been principally studied in relation to amyloid-beta pathology but, comparatively, little is known about inflammatory processes associated to tau pathology. In the hippocampus of AD patients, where tau pathology is more prominent than amyloid-beta pathology, a microglial degenerative process has been reported. In this work, we have directly compared the microglial response in two different transgenic tau mouse models: ThyTau22 and P301S. Surprisingly, these two models showed important differences in the microglial profile and tau pathology. Where ThyTau22 hippocampus manifested mild microglial activation, P301S mice exhibited a strong microglial response in parallel with high phospho-tau accumulation. This differential phospho-tau expression could account for the different microglial response in these two tau strains. However, soluble (S1) fractions from ThyTau22 hippocampus presented relatively high content of soluble phospho-tau (AT8-positive) and were highly toxic for microglial cells in vitro, whereas the correspondent S1 fractions from P301S mice displayed low soluble phosphotau levels and were not toxic for microglial cells. Therefore, not only the expression levels but the aggregation of phospho-tau should differ between both models. In fact, most of tau forms in the P301S mice were aggregated and, in consequence, forming insoluble tau species.We conclude that different factors as tau mutations, accumulation, phosphorylation, and/or aggregation could account for the distinct microglial responses observed in these two tau models. For this reason, deciphering the molecular nature of toxic tau species for microglial cells might be a promising therapeutic approach in order to restore the deficient immunological protection observed in AD hippocampus.CIBERNEDJunta de Andalucía. Consejería de Economía, Innovación, Ciencia y Empleo CTS-2035Fundación Tatiana Pérez de Guzmán el BuenoMinisterio de Ciencia, Innovación y UniversidadesInstituto de Salud Carlos III. Fondo de Investigación Sanitaria. PI15/00957 PI15/00796Fondo Europeo de Desarrollo Regional PI15/00957 PI15/0079

Finding the principal points of a random variable

The p-principal points of a random variable X with finite second moment are those p points in R minimizing the expected squared distance from X to the closest point. Although the determination of principal points involves in general the resolution of a multiextremal optimization problem, existing procedures in the literature provide just a local optimum. In this paper we show that standard Global Optimization techniques can be applied.Ministerio de Ciencia y Tecnologí

Formation of phenotypic lineages in Salmonella enterica by a pleiotropic fimbrial switch

The std locus of Salmonella enterica, an operon acquired by horizontal transfer, encodes fimbriae that permit adhesion to epithelial cells in the large intestine. Expression of the std operon is bistable, yielding a major subpopulation of StdOFF cells (99.7%) and a minor subpopulation of StdON cells (0.3%). In addition to fimbrial proteins, the std operon encodes two proteins, StdE and StdF, that have DNA binding capacity and control transcription of loci involved in flagellar synthesis, chemotaxis, virulence, conjugal transfer, biofilm formation, and other cellular functions. As a consequence of StdEF pleiotropic transcriptional control, StdON and StdOFF subpopulations may differ not only in the presence or absence of Std fimbriae but also in additional phenotypic traits. Separation of StdOFF and StdON lineages by cell sorting confirms the occurrence of lineage-specific features. Formation of StdOFF and StdON lineages may thus be viewed as a rudimentary bacterial differentiation program