Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant

The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membraneThis work was supported by the Spanish Ministerio de Ciencia e Innovación Grant BFU2008-0428

A novel Salmonella periplasmic protein controlling cell wall homeostasis and virulence

Horizontal gene transfer has shaped the evolution of Salmonella enterica as pathogen. Some functions acquired by this mechanism include enzymes involved in peptidoglycan (PG) synthesis and remodeling. Here, we report a novel serovar Typhimurium protein that is absent in non-pathogenic bacteria and bears a LprI functional domain, first reported in a Mycobacterium tuberculosis lipoprotein conferring lysozyme resistance. Based on the presence of such domain, we hypothesized a role of this S. Typhimurium protein in PG metabolism. This protein, which we named ScwA for Salmonella cell wall-related regulator-A, controls positively the levels of the murein lytic transglycosylase MltD. In addition, the levels of other enzymes that cleave bonds in the PG lattice were affected in a mutant lacking ScwA, including a soluble lytic tranglycosylase (Slt), the amidase AmiC, and a few endo- and carboxypeptidases (NlpC, PBP4, and AmpH). The scwA gene has lower G+C content than the genomic average (43.1 vs. 52.2%), supporting acquisition by horizontal transfer. ScwA is located in the periplasm, stabilized by two disulfide bridges, produced preferentially in stationary phase and down-regulated following entry of the pathogen into eukaryotic cells. ScwA deficiency, however, results in a hypervirulent phenotype in the murine typhoid model. Based on these findings, we conclude that ScwA may be exploited by S. Typhimurium to ensure cell envelope homeostasis along the infection and to prevent host overt damage. This role could be accomplished by controlling the production or stability of a reduced number of peptidoglycan hydrolases whose activities result in the release of PG fragments.Ministry of Science and Innovatio

Affinity of cefotiam for the alternative penicillin binding protein PBP3SAL used by Salmonella inside host eukaryotic cells

Background: Following the invasion of eukaryotic cells, Salmonella enterica serovar Typhimurium replaces PBP2/PBP3, main targets of β-lactam antibiotics, with PBP2SAL/PBP3SAL, two homologue peptidoglycan synthases absent in Escherichia coli. PBP3SAL promotes pathogen cell division in acidic environments independently of PBP3 and shows low affinity for β-lactams that bind to PBP3 such as aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefalotin. Objectives: To find compounds with high affinity for PBP3SAL to control Salmonella intracellular infections. Methods: An S. Typhimurium ΔPBP3 mutant that divides using PBP3SAL and its parental wild-type strain, were exposed to a library of 1520 approved drugs in acidified (pH 4.6) nutrient-rich LB medium. Changes in optical density associated with cell filamentation, a read-out of blockage in cell division, were monitored. Compounds causing filamentation in the ΔPBP3 mutant but not in wild-type strain-the latter strain expressing both PBP3 and PBP3SAL in LB pH 4.6-were selected for further study. The bactericidal effect due to PBP3SAL inhibition was evaluated in vitro using a bacterial infection model of cultured fibroblasts. Results: The cephalosporin cefotiam exhibited higher affinity for PBP3SAL than for PBP3 in bacteria growing in acidified LB pH 4.6 medium. Cefotiam also proved to be effective against intracellular Salmonella in a PBP3SAL-dependent manner. Conversely, cefuroxime, which has higher affinity for PBP3, showed decreased effectiveness in killing intracellular Salmonella. Conclusions: Antibiotics with affinity for PBP3SAL, like the cephalosporin cefotiam, have therapeutic value for treating Salmonella intracellular infections.This work was supported by grants PDC2021-121094-I00/10.13039/501100011033 (to F.G-d.P.) from the Spanish Ministry of Science and Innovation and the Next Generation European Union EU/PRTR Program, and PID2020-114546RB (to O.Z.) from the Spanish Ministry of Science and Innovation.S

Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries

Ajuts: Departamento Administrativo de Ciencias, Tecnología e Innovación (Colciencias), República de Colombia; Convocatoria 489 - 2009, Código 657048925406, Contrato de financiación RC. 427 - 2009 Colciencias - CorpoGen; Programa de Asistencias Graduadas de Universidad de los Andes, Bogotá, Colombia; i Programa Jóvenes Investigadores de ColcienciasBACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been develope

Evaluación de dos Métodos para el Aprovechamiento Turístico en Áreas Naturales Protegidas

El objetivo general fue realizar un análisis crítico del método de Límites de Cambio Aceptable y Capacidad de Carga Turística en áreas naturales protegidas, con énfasis en la propuesta de Cifuentes et al. (1999) por ser el instrumento más aplicado en América Latina. Se realizó una revisión bibliográfica de artículos científicos referidos al objeto de estudio. Como resultados, se determina que el método Cifuentes se enfoca en aspectos lineales, uni-dimensionales y parciales, no considera la homeóstasis y omite la zonificación previa, lo cual facilita su operacionalización en las áreas naturales protegidas pero genera valores heurísticos. Se sugiere fortalecer el método con la inclusión de aspectos sociales y económicos para integrar las tres dimensiones de la sustentabilidad, así como incorporar la zona de influencia al área natural protegida dentro del análisis integrador en la planificación turística

Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries

BACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. CONCLUSIONS: Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been developed

Non-volatile tuning of normally-on and off states of deep depletion ZrO2/O-terminated high voltage diamond MOSFET

Based on the stability of the deep depletion regime in diamond and the outstanding properties of this promising material for its use in power devices, p-channel deep depletion metal oxide semiconductor field effect transistors were fabricated on a (001) Ib nitrogen-doped high pressure high temperature diamond substrate. Taking advantage of the new concept of the non-volatile diamond-based photo switch, it is demonstrated that it is possible to tune the normally-on and normally-off states of the transistor by configuring the pn-junction space charge region. The devices under study was designed following an interdigital-like and a circular-like architectures presenting low threshold voltages (between 3 V and −3 V), an on/off ratio of 107 and a critical electric field numerically assessed of 9 MV.cm−1 at room temperature. This new degree of freedom opens the route for diamond based power electronics.8 página

Iron deprivation enhances transcriptional responses to in vitro growth arrest of Mycobacterium tuberculosis

The establishment of Mycobacterium tuberculosis (Mtb) long-term infection in vivo depends on several factors, one of which is the availability of key nutrients such as iron (Fe). The relation between Fe deprivation inside and outside the granuloma, and the capacity of Mtb to accumulate lipids and persist in the absence of growth is not well understood. In this context, current knowledge of how Mtb modifies its lipid composition in response to growth arrest, depending on iron availability, is scarce. To shed light on these matters, in this work we compare genome-wide transcriptomic and lipidomic profiles of Mtb at exponential and stationary growth phases using cultures with glycerol as a carbon source, in the presence or absence of iron. As a result, we found that transcriptomic responses to growth arrest, considered as the transition from exponential to stationary phase, are iron dependent for as many as 714 genes (iron-growth interaction contrast, FDR <0.05), and that, in a majority of these genes, iron deprivation enhances the magnitude of the transcriptional responses to growth arrest in either direction. On the one hand, genes whose upregulation upon growth arrest is enhanced by iron deprivation were enriched in functional terms related to homeostasis of ion metals, and responses to several stressful cues considered cardinal features of the intracellular environment. On the other hand, genes showing negative responses to growth arrest that are stronger in iron-poor medium were enriched in energy production processes (TCA cycle, NADH dehydrogenation and cellular respiration), and key controllers of ribosomal activity shut-down, such as the T/A system mazE6/F6. Despite of these findings, a main component of the cell envelope, lipid phthiocerol dimycocerosate (PDIM), was not detected in the stationary phase regardless of iron availability, suggesting that lipid changes during Mtb adaptation to non-dividing phenotypes appear to be iron-independent. Taken together, our results indicate that environmental iron levels act as a key modulator of the intensity of the transcriptional adaptations that take place in the bacterium upon its transition between dividing and dormant-like phenotypes in vitro

Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections