Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Structural snapshots of OxyR reveal the peroxidatic mechanism of H2O2 sensing.

Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2 One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2 In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.

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.

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.

Resistance is futile: targeting multidrug-resistant bacteria with de novo Cys-rich cyclic polypeptides.

The search for novel antimicrobial agents to combat microbial pathogens is intensifying in response to rapid drug resistance development to current antibiotic therapeutics. The use of disulfide-rich head-to-tail cyclized polypeptides as molecular frameworks for designing a new type of peptide antibiotics is gaining increasing attention among the scientific community and the pharmaceutical industry. The use of macrocyclic peptides, further constrained by the presence of several disulfide bonds, makes these peptide frameworks remarkably more stable to thermal, biological, and chemical degradation showing better activities when compared to their linear analogs. Many of these novel peptide scaffolds have been shown to have a high tolerance to sequence variability in those residues not involved in disulfide bonds, able to cross biological membranes, and efficiently target complex biomolecular interactions. Hence, these unique properties make the use of these scaffolds ideal for many biotechnological applications, including the design of novel peptide antibiotics. This article provides an overview of the new developments in the use of several disulfide-rich cyclic polypeptides, including cyclotides, θ-defensins, and sunflower trypsin inhibitor peptides, among others, in the development of novel antimicrobial peptides against multidrug-resistant bacteria.

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.

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.

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.

Microbial Production of Ethanol From Sludge Derived From an Urban Wastewater Treatment Plant.

A collection of lipase-producing microorganisms was isolated from sludge derived from an urban wastewater treatment plant. The microorganisms with the highest levels of lipase activity were selected in order to use triglycerides present in the sludge effectively and were then transformed with pdc:adhB genes for the production of ethanol. The transgenic strains showed high growth rates in diluted sludge and produced lipase protein in order to utilize fat present in the sludge, which provides an abundant source of carbon. Using sludge derived from treated wastewater as nutrient source, ethanol was produced by certain transgenic species belonging to the genera Proteus. Different forms of sludge were tested for maximal ethanol production, with dehydrated sludge being found to produce the best performance.