Corynebacterium glutamicum as a model bacterium for the bioremediation of arsenic

Arsenic is an extremely toxic metalloid that, when present in high concentrations, severely threatens the biota and human health. Arsenic contamination of soil, water, and air is a global growing environmental problem due to leaching from geological formations, the burning of fossil fuels, wastes generated by the gold mining industry present in uncontrolled landfills, and improper agriculture or medical uses. Unlike organic contaminants, which are degraded into harmless chemical species, metals and metalloids cannot be destroyed, but they can be immobilized or transformed into less toxic forms. The ubiquity of arsenic in the environment has led to the evolution in microbes of arsenic defense mechanisms. The most common of these mechanisms is based on the presence of the arsenic resistance operon (ars), which codes for: (i) a regulatory protein, ArsR; (ii) an arsenite permease, ArsB; and (iii) an enzyme involved in arsenate reduction, ArsC. Corynebacterium glutamicum, which is used for the industrial production of amino acids and nucleotides, is one of the most arsenic-resistant microorganisms described to date (up to 12 mM arsenite and >400 mM arseniate). Analysis of the C. glutamicum genome revealed the presence of two complete ars operons (ars1 and ars2) comprising the typical three-gene structure arsRBC, with an extra arsC1´ located downstream from arsC1 (ars1 operon), and two orphan genes (arsB3 and arsC4). The involvement of both ars operons in arsenic resistance in C. glutamicum was confirmed by disruption and amplification of those genes. The strains obtained were resistant to up to 60 mM arsenite, one of the highest levels of bacterial resistance to arsenite so far described. Using tools for the genetic manipulation of C. glutamicum that were developed in our laboratory, we are attempting to obtain C. glutamicum mutant strains able to remove arsenic from contaminated water. [Int Microbiol 2006; 9(3):207-215

Cytoskeletal Proteins of Actinobacteria

Although bacteria are considered the simplest life forms, we are now slowly unraveling their cellular complexity. Surprisingly, not only do bacterial cells have a cytoskeleton but also the building blocks are not very different from the cytoskeleton that our own cells use to grow and divide. Nonetheless, despite important advances in our understanding of the basic physiology of certain bacterial models, little is known about Actinobacteria, an ancient group of Eubacteria. Here we review current knowledge on the cytoskeletal elements required for bacterial cell growth and cell division, focusing on actinobacterial genera such as Mycobacterium, Corynebacterium, and Streptomyces. These include some of the deadliest pathogens on earth but also some of the most prolific producers of antibiotics and antitumorals

TMEM59 defines a novel ATG16L1-binding motif that promotes local activation of LC3

Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD-repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1-binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.This work was funded by grants from the Ministerio de Ciencia e Innovación of the Spanish Government (Refs SAF2008‐00350 and SAF2011‐23714), Fundación Solórzano, Junta de Castilla y León (Consejería de Educación, Ref. CSI001A10‐2, and Consejería de Sanidad) and Consejo Superior de Investigaciones Científicas (CSIC; Ref. 200720I026). Additional funding comes from the FEDER programme of the European Union. EB is a graduate student funded by a predoctoral fellowship from the FPU programme (Ministerio de Educación, MEC, Spanish Government). ML is funded by JAE‐Doc and Juan de la Cierva postdoctoral contracts (MEC and Social European Fund of the European Union, 2007–2013). AF is funded by a long‐term EMBO postdoctoral fellowship and a Juan de la Cierva contract. KP is a graduate student funded by an FPI fellowship (MEC).Peer Reviewe

Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi

[EN] Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectivesS

Intracellular Staphylococcus aureus modulates host central carbon metabolism to activate autophagy

Staphylococcus aureus is a facultative intracellular pathogen that invades and replicates within many types of phagocytic and nonphagocytic cells. During intracellular infection, S. aureus is capable of subverting xenophagy and escaping to the cytosol of the host cell. Furthermore, drug-induced autophagy facilitates the intracellular replication of S. aureus, but the reasons behind this are unclear. Here, we have studied the host central carbon metabolism during S. aureus intracellular infection. We found extensive metabolic rerouting and detected several distinct metabolic changes that suggested starvation-induced autophagic flux in infected cells. These changes included increased uptake but lower intracellular levels of glucose and low abundance of several essential amino acids, as well as markedly upregulated glutaminolysis. Furthermore, we show that AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) phosphorylation levels are significantly increased in infected cells. Interestingly, while autophagy was activated in response to S. aureus invasion, most of the autophagosomes detected in infected cells did not contain bacteria, suggesting that S. aureus induces the autophagic flux during cell invasion for energy generation and nutrient scavenging. Accordingly, AMPK inhibition halted S. aureus intracellular proliferation

Understanding microRNAs in the Context of Infection to Find New Treatments against Human Bacterial Pathogens

[EN] The development of RNA-based anti-infectives has gained interest with the successful application of mRNA-based vaccines. Small RNAs are molecules of RNA of <200 nucleotides in length that may control the expression of specific genes. Small RNAs include small interference RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), or microRNAs (miRNAs). Notably, the role of miRNAs on the post-transcriptional regulation of gene expression has been studied in detail in the context of cancer and many other genetic diseases. However, it is also becoming apparent that some human miRNAs possess important antimicrobial roles by silencing host genes essential for the progress of bacterial or viral infections. Therefore, their potential use as novel antimicrobial therapies has gained interest during the last decade. The challenges of the transport and delivery of miRNAs to target cells are important, but recent research with exosomes is overcoming the limitations in RNA-cellular uptake, avoiding their degradation. Therefore, in this review, we have summarised the latest developments in the exosomal delivery of miRNA-based therapies, which may soon be another complementary treatment to pathogen-targeted antibiotics that could help solve the problem caused by multidrug-resistant bacteriaSIWe thank the Junta de Castilla y León (Spain) for funding our research work on microRNAs, grant number LE044P20. A.M. is supported with a postdoctoral fellowship “Margarita Salas”. M.L. is the recipient of a “Beatriz Galindo” grant (Ref. BEAGAL18/00068 - BGP18/00033

Repetitive Exposure of IL-17 Into the Murine Air Pouch Favors the Recruitment of Inflammatory Monocytes and the Release of IL-16 and TREM-1 in the Inflammatory Fluids

The infiltration of Th17 cells in tissues and organs during the development of many autoimmune diseases is considered a key step toward the establishment of chronic inflammation. Indeed, the localized and prolonged release of IL-17 in specific tissues has been associated with an increased severity of the inflammatory response that remains sustained over time. The cellular and molecular mechanisms behind these effects are far from being clear. In this study we investigated the effects of two repetitive administration of recombinant IL-17 into the murine air pouch to simulate a scenario where IL-17 is released over time in a pre-inflamed tissue. Consistent with our previous observations, mice receiving a single dose of IL-17 showed a transitory influx of neutrophils into the air pouch that peaked at 24 h and declined at 48 h. Conversely, mice receiving a double dose of the cytokine—one at time 0 and the second after 24 h—showed a more dramatic inflammatory response with almost 2-fold increase in the number of infiltrated leukocytes and significant higher levels of TNF-α and IL-6 in the inflammatory fluids. Further analysis of the exacerbated inflammatory response of double-injected IL-17 mice showed a unique cellular and biochemical profile with inflammatory monocytes as the second main population emigrating to the pouch and IL-16 and TREM-1 as the most upregulated cytokines found in the inflammatory fluids. Most interestingly, mice receiving a double injection of IL-1β did not show any change in the cellular or biochemical inflammatory response compared to those receiving a single injection or just vehicle. Collectively these results shed some light on the function of IL-17 as pro-inflammatory cytokine and provide possible novel ways to target therapeutically the pathogenic effects of IL-17 in autoimmune conditions

Aprendizaje-Servicio en el entorno rural de León

[ES] A través del grupo de Innovación Docente BIOMETAC, nace esta experiencia de ApS para promover en los estudiantes de los grados en Biología, Biotecnología y Ciencias Ambientales de la ULE, la iniciativa cívica de participación en la sociedad. El objetivo general del proyecto es que el alumnado aplique sus conocimientos y habilidades para sensibilizar y dinamizar a la población de varios municipios rurales de León frente a retos de la Agenda 2030 asociados a la salud y la sostenibilidad ambiental. Se establecieron 3 grupos de trabajo, cada uno integrado por 12 estudiantes de los tres grados, que trabajan en 3 municipios rurales de la provincia de León: Cistierna, Truchas y Villablino, con diversas problemáticas en el ámbito de la salud y medioambiente. El Proyecto se realiza en tres fases: Preparación, Ejecución y Evaluación y Difusión. Durante la primera fase (curso 2021/22), se realizaron reuniones con el alumnado para guiarlos en el diagnóstico de cada municipio. Los datos obtenidos permitieron detectar los problemas que más preocupaban a la población de cada municipio. En una segunda fase (curso 2022/23), los alumnos profundizarán en las distintas problemáticas mediante actuaciones propuestas por ellos mismos y presentarán sus conclusiones en cada municipio. Esta experiencia pretende que los estudiantes pongan los conocimientos y las competencias adquiridas al servicio de comunidades rurales, trabajando directamente sobre varios Objetivos de Desarrollo Sostenible (ODS), mejorando su capacidad de iniciativa, autonomía en la organización del trabajo, y responsabilidad para la resolución de problemas

The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi