The extracellular thioredoxin Etrx3 is required for macrophage infection in Rhodococcus equi.

Rhodococcus equi is an intracellular veterinary pathogen that is becoming resistant to current antibiotherapy. Genes involved in preserving redox homeostasis could be promising targets for the development of novel anti-infectives. Here, we studied the role of an extracellular thioredoxin (Etrx3/REQ_13520) in the resistance to phagocytosis. An etrx3-null mutant strain was unable to survive within macrophages, whereas the complementation with the etrx3 gene restored its intracellular survival rate. In addition, the deletion of etrx3 conferred to R. equi a high susceptibility to sodium hypochlorite. Our results suggest that Etrx3 is essential for the resistance of R. equi to specific oxidative agents.

Acetaminophen cytotoxicity in HepG2 cells is associated with a decoupling of glycolysis from the TCA cycle, loss of NADPH production, and suppression of anabolism.

Acetaminophen (APAP) is one of the most commonly used analgesics worldwide, and overdoses are associated with lactic acidosis, hepatocyte toxicity, and acute liver failure due to oxidative stress and mitochondrial dysfunction. Hepatoma cell lines typically lack the CYP450 activity to generate the reactive metabolite of APAP observed in vivo, but are still subject to APAP cytotoxicity. In this study, we employed metabolic profiling and isotope labelling approaches to investigate the metabolic impact of acute exposure to cytotoxic doses of APAP on the widely used HepG2 cell model. We found that APAP exposure leads to limited cellular death and substantial growth inhibition. Metabolically, we observed an up-regulation of glycolysis and lactate production with a concomitant reduction in carbon from glucose entering the pentose-phosphate pathway and the TCA cycle. This was accompanied by a depletion of cellular NADPH and a reduction in the de novo synthesis of fatty acids and the amino acids serine and glycine. These events were not associated with lower reduced glutathione levels and no glutathione conjugates were seen in cell extracts. Co-treatment with a specific inhibitor of the lactate/H+ transporter MCT1, AZD3965, led to increased apoptosis in APAP-treated cells, suggesting that lactate accumulation could be a cause of cell death in this model. In conclusion, we show that APAP toxicity in HepG2 cells is largely independent of oxidative stress, and is linked instead to a decoupling of glycolysis from the TCA cycle, lactic acidosis, reduced NADPH production, and subsequent suppression of the anabolic pathways required for rapid growth.

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.

The Arsenic Detoxification System in Corynebacteria: Basis and Application for Bioremediation and Redox Control.

Arsenic (As) is widespread in the environment and highly toxic. It has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. To survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (AsV) to arsenite (AsIII) by arsenate reductases, which is then extruded by arsenite permeases. One of these Gram-positive bacteria, Corynebacterium glutamicum, the workhorse of biotechnological research, is also resistant to arsenic. To sanitize contaminated soils and waters, C. glutamicum strains were modified to work as arsenic "biocontainers." Two chromosomally encoded ars operons (ars1 and ars2) are responsible for As resistance. The genes within these operons encode for metalloregulatory proteins (ArsR1/R2), arsenite permeases (Acr3-1/-2), and arsenate reductases (ArsC1/C2/C1'). ArsC1/C2 arsenate reductases are coupled to the low molecular weight thiol mycothiol (MSH) and to the recently discovered mycoredoxin-1 (Mrx-1) present in most Actinobacteria. This MSH/Mrx-1 redox system protects cells against different forms of stress, including reactive oxygen species (ROS), metals, and antibiotics. ROS can modify functional sulfur cysteines by oxidizing the thiol (-SH) to a sulfenic acid (-SOH). These oxidation-sensitive protein cysteine thiols are redox regulated by the MSH/Mrx-1 couple in Corynebacterium and Mycobacterium. In summary, the molecular mechanisms involved in arsenic resistance system in C. glutamicum have paved the way for understanding the cellular response against oxidative stress in Actinobacteria.

BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α.

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.

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.

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

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 bacteria.

Phosphorylation of a novel cytoskeletal protein (RsmP) regulates rod-shaped morphology in Corynebacterium glutamicum.

Corynebacteria grow by wall extension at the cell poles, with DivIVA being an essential protein orchestrating cell elongation and morphogenesis. DivIVA is considered a scaffolding protein able to recruit other proteins and enzymes involved in polar peptidoglycan biosynthesis. Partial depletion of DivIVA induced overexpression of cg3264, a previously uncharacterized gene that encodes a novel coiled coil-rich protein specific for corynebacteria and a few other actinomycetes. By partial depletion and overexpression of Cg3264, we demonstrated that this protein is an essential cytoskeletal element needed for maintenance of the rod-shaped morphology of Corynebacterium glutamicum, and it was therefore renamed RsmP (rod-shaped morphology protein). RsmP forms long polymers in vitro in the absence of any cofactors, thus resembling eukaryotic intermediate filaments. We also investigated whether RsmP could be regulated post-translationally by phosphorylation, like eukaryotic intermediate filaments. RsmP was phosphorylated in vitro by the PknA protein kinase and to a lesser extent by PknL. A mass spectrometric analysis indicated that phosphorylation exclusively occurred on a serine (Ser-6) and two threonine (Thr-168 and Thr-211) residues. We confirmed that mutagenesis to alanine (phosphoablative protein) totally abolished PknA-dependent phosphorylation of RsmP. Interestingly, when the three residues were converted to aspartic acid, the phosphomimetic protein accumulated at the cell poles instead of making filaments along the cell, as observed for the native or phosphoablative RsmP proteins, indicating that phosphorylation of RsmP is necessary for directing cell growth at the cell poles.

Novel Treatments and Preventative Strategies Against Food-Poisoning Caused by Staphylococcal Species.

Staphylococcal infections are a widespread cause of disease in humans. In particular, S. aureus is a major causative agent of infection in clinical medicine. In addition, these bacteria can produce a high number of staphylococcal enterotoxins (SE) that may cause food intoxications. Apart from S. aureus, many coagulase-negative Staphylococcus spp. could be the source of food contamination. Thus, there is an active research work focused on developing novel preventative interventions based on food supplements to reduce the impact of staphylococcal food poisoning. Interestingly, many plant-derived compounds, such as polyphenols, flavonoids, or terpenoids, show significant antimicrobial activity against staphylococci, and therefore these compounds could be crucial to reduce the incidence of food intoxication in humans. Here, we reviewed the most promising strategies developed to prevent staphylococcal food poisoning.

Novel Methods to Identify Oxidative Stress-Producing Antibiotics.

Antibiotherapy is the main therapeutic strategy in the fight against bacterial pathogens. However, the misuse of antimicrobials has led to the appearance of antimicrobial-resistant strains. The rate at which we isolate multidrug-resistant bacteria is now much faster than the discovery rate of new antimicrobials. Therefore, the repurposing of approved drugs against multidrug-resistant bacteria is a very promising strategy to find new therapies against these pathogens. Some antibiotics generate oxidative stress as part of their mechanism of action. We have recently applied different methods to find new oxidative stress-producing antimicrobials with synergistic action against intracellular pathogens. Here, we detail several procedures that could be used to identify oxidative stress-producing antimicrobials with a synergistic mechanism of action.

Novel Treatments against Mycobacterium tuberculosis Based on Drug Repurposing.

Tuberculosis is the leading cause of death, worldwide, due to a bacterial pathogen. This respiratory disease is caused by the intracellular pathogen Mycobacterium tuberculosis and produces 1.5 million deaths every year. The incidence of tuberculosis has decreased during the last decade, but the emergence of MultiDrug-Resistant (MDR-TB) and Extensively Drug-Resistant (XDR-TB) strains of M. tuberculosis is generating a new health alarm. Therefore, the development of novel therapies based on repurposed drugs against MDR-TB and XDR-TB have recently gathered significant interest. Recent evidence, focused on the role of host molecular factors on M. tuberculosis intracellular survival, allowed the identification of new host-directed therapies. Interestingly, the mechanism of action of many of these therapies is linked to the activation of autophagy (e.g., nitazoxanide or imatinib) and other well-known molecular pathways such as apoptosis (e.g., cisplatin and calycopterin). Here, we review the latest developments on the identification of novel antimicrobials against tuberculosis (including avermectins, eltrombopag, or fluvastatin), new host-targeting therapies (e.g., corticoids, fosfamatinib or carfilzomib) and the host molecular factors required for a mycobacterial infection that could be promising targets for future drug development.

Alternative Anti-Infective Treatments to Traditional Antibiotherapy against Staphylococcal Veterinary Pathogens.

The genus Staphylococcus encompasses many species that may be pathogenic to both humans and farm animals. These bacteria have the potential to acquire multiple resistant traits to the antimicrobials currently used in the veterinary or medical settings. These pathogens may commonly cause zoonoses, and the infections they cause are becoming difficult to treat due to antimicrobial resistance. Therefore, the development of novel alternative treatments to traditional antibiotherapy has gained interest in recent years. Here, we reviewed the most promising therapeutic strategies developed to control staphylococcal infections in the veterinary field to overcome antibiotic resistance.

Oxidative Stress-Generating Antimicrobials, a Novel Strategy to Overcome Antibacterial Resistance.

Antimicrobial resistance is becoming one of the most important human health issues. Accordingly, the research focused on finding new antibiotherapeutic strategies is again becoming a priority for governments and major funding bodies. The development of treatments based on the generation of oxidative stress with the aim to disrupt the redox defenses of bacterial pathogens is an important strategy that has gained interest in recent years. This approach is allowing the identification of antimicrobials with repurposing potential that could be part of combinatorial chemotherapies designed to treat infections caused by recalcitrant bacterial pathogens. In addition, there have been important advances in the identification of novel plant and bacterial secondary metabolites that may generate oxidative stress as part of their antibacterial mechanism of action. Here, we revised the current status of this emerging field, focusing in particular on novel oxidative stress-generating compounds with the potential to treat infections caused by intracellular bacterial pathogens.

A Novel Screening Strategy Reveals ROS-Generating Antimicrobials That Act Synergistically against the Intracellular Veterinary Pathogen Rhodococcus equi.

: Rhodococcus equi is a facultative intracellular pathogen that causes infections in foals and many other animals such as pigs, cattle, sheep, and goats. Antibiotic resistance is rapidly rising in horse farms, which makes ineffective current antibiotic treatments based on a combination of macrolides and rifampicin. Therefore, new therapeutic strategies are urgently needed to treat R. equi infections caused by antimicrobial resistant strains. Here, we employed a R. equi mycoredoxin-null mutant strain highly susceptible to oxidative stress to screen for novel ROS-generating antibiotics. Then, we used the well-characterized Mrx1-roGFP2 biosensor to confirm the redox stress generated by the most promising antimicrobial agents identified in our screening. Our results suggest that different combinations of antibacterial compounds that elicit oxidative stress are promising anti-infective strategies against R. equi. In particular, the combination of macrolides with ROS-generating antimicrobial compounds such as norfloxacin act synergistically to produce a potent antibacterial effect against R. equi. Therefore, our screening approach could be applied to identify novel ROS-inspired therapeutic strategies against intracellular pathogens.

Comparative Evaluation of the Antimicrobial and Mucus Induction Properties of Selected Bacillus Strains against Enterotoxigenic Escherichia coli.

Probiotics have been shown to bind to host receptors, which are important for pathogen adhesion and induce the host's production of defence factors. They can activate the goblet-cell-derived production of mucins, a major component of the mucus layer and a physical barrier participating in limiting the proximity of microorganisms to the epithelial layer. In the last decade, Bacillus spp. strains have gained interest in human and animal health due to their tolerance and stability under gastrointestinal tract conditions. Moreover, Bacillus spp. strains can also produce various antimicrobial peptides that can support their use as commercial probiotic supplements and functional foods. The present study aimed to evaluate and determine the ability of selected Bacillus spp. strains to inhibit the growth of enterotoxigenic Escherichia coli (ETEC) F4 and to reduce binding of ETEC F4 to HT29-16E (mucus-secreting and goblet-like) human intestinal cells. Moreover, mucus production in the HT29 cells in the presence of the Bacillus spp. strains was quantified by ELISA. Bacillus spp. strains (CHCC 15076, CHCC 15516, CHCC 15541, and CHCC 16872) significantly inhibited the growth of ETEC F4. Moreover, the ability of the probiotic Bacillus spp. strains to stimulate mucin release was highly strain dependent. The treatment with Bacillus subtilis CHCC 15541 resulted in a significant increase of both MUC2 and MUC3 in HT29-16E cells. Therefore, this strain could be an up-and-coming candidate for developing commercial probiotic supplements to prevent infections caused by ETEC F4 and, potentially, other pathogens.

Host-Targeted Therapeutics against Multidrug Resistant Intracellular Staphylococcus aureus.

Staphylococcus aureus is a facultative intracellular pathogen that invades and replicates within many types of human cells. S. aureus has shown to rapidly overcome traditional antibiotherapy by developing multidrug resistance. Furthermore, intracellular S. aureus is protected from the last-resort antibiotics-vancomycin, daptomycin, and linezolid-as they are unable to achieve plasma concentrations sufficient for intracellular killing. Therefore, there is an urgent need to develop novel anti-infective therapies against S. aureus infections. Here, we review the current state of the field and highlight the exploitation of host-directed approaches as a promising strategy going forward.

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

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-infectives.

Identification of novel targets for host-directed therapeutics against intracellular Staphylococcus aureus.

During patient colonization, Staphylococcus aureus is able to invade and proliferate within human cells to evade the immune system and last resort drugs such as vancomycin. Hijacking specific host molecular factors and/or pathways is necessary for pathogens to successfully establish an intracellular infection. In this study, we employed an unbiased shRNA screening coupled with ultra-fast sequencing to screen 16,000 human genes during S. aureus infection and we identified several host genes important for this intracellular pathogen. In addition, we interrogated our screening results to find novel host-targeted therapeutics against intracellular S. aureus. We found that silencing the human gene TRAM2 resulted in a significant reduction of intracellular bacterial load while host cell viability was restored, showing its importance during intracellular infection. Furthermore, TRAM2 is an interactive partner of the endoplasmic reticulum SERCA pumps and treatment with the SERCA-inhibitor Thapsigargin halted intracellular MRSA survival. Our results suggest that Thapsigargin could be repurposed to tackle S. aureus host cell infection in combination with conventional antibiotics.

Intracellular Staphylococcus aureus Elicits the Production of Host Very Long-Chain Saturated Fatty Acids with Antimicrobial Activity.

As a facultative intracellular pathogen, Staphylococcus aureus is able to invade and proliferate within many types of mammalian cells. Intracellular bacterial replication relies on host nutrient supplies and, therefore, cell metabolism is closely bound to intracellular infection. Here, we investigated how S. aureus invasion affects the host membrane-bound fatty acids. We quantified the relative levels of fatty acids and their labelling pattern after intracellular infection by gas chromatography-mass spectrometry (GC-MS). Interestingly, we observed that the levels of three host fatty acids-docosanoic, eicosanoic and palmitic acids-were significantly increased in response to intracellular S. aureus infection. Accordingly, labelling carbon distribution was also affected in infected cells, in comparison to the uninfected control. In addition, treatment of HeLa cells with these three fatty acids showed a cytoprotective role by directly reducing S. aureus growth.