The glutathione import system satisfies the Staphylococcus aureus nutrient sulfur requirement and promotes interspecies competition.

Sulfur is an indispensable element for bacterial proliferation. Prior studies demonstrated that the human pathogen Staphylococcus aureus utilizes glutathione (GSH) as a source of nutrient sulfur; however, mechanisms of GSH acquisition are not defined. Here, we identify a five-gene locus comprising a putative ABC-transporter and predicted γ-glutamyl transpeptidase (ggt) that promotes S. aureus proliferation in medium supplemented with either reduced or oxidized GSH (GSSG) as the sole source of nutrient sulfur. Based on these phenotypes, we name this transporter operon the glutathione import system (gisABCD). Ggt is encoded within the gisBCD operon, and we show that the enzyme is capable of liberating glutamate using either GSH or GSSG as substrates, demonstrating it is a bona fide γ-glutamyl transpeptidase. We also determine that Ggt is expressed in the cytoplasm, representing only the second example of cytoplasmic Ggt localization, the other being Neisseria meningitidis. Bioinformatic analyses revealed that Staphylococcus species closely related to S. aureus encode GisABCD-Ggt homologs. However, homologous systems were not detected in Staphylococcus epidermidis. Consequently, we establish that GisABCD-Ggt provides a competitive advantage for S. aureus over S. epidermidis in a GSH- and GSSG-dependent manner. Overall, this study describes the discovery of a nutrient sulfur acquisition system in S. aureus that targets GSSG in addition to GSH and promotes competition against other staphylococci commonly associated with the human microbiota.

Crucial Role for Lipoteichoic Acid Assembly in the Metabolic Versatility and Antibiotic Resistance of Staphylococcus aureus.

Staphylococcus aureus is a public health threat due to the prevalence of antibiotic resistance and the capacity of this organism to infect numerous organs in vertebrates. To generate energy needed to proliferate within tissues, S. aureus transitions between aerobic respiration and fermentation. Fermentation results in a distinct colony morphology called the small-colony variant (SCV) due to decreased membrane potential and ATP production. These traits promote increased resistance to aminoglycoside antibiotics. Consequently, SCVs are associated with persistent infections. We hypothesize that dedicated physiological pathways support fermentative growth of S. aureus that represent potential targets for treatment of resistant infections. Lipoteichoic acid (LTA) is an essential component of the Gram-positive cell envelope that functions to maintain ion homeostasis, resist osmotic stress, and regulate autolytic activity. Previous studies revealed that perturbation of LTA reduces viability of metabolically restricted S. aureus, but the mechanism by which LTA supports S. aureus metabolic versatility is unknown. Though LTA is essential, the enzyme that synthesizes the modified lipid anchor, YpfP, is dispensable. However, ypfP mutants produce altered LTA, leading to elongation of the polymer and decreased cell association. We demonstrate that viability of ypfP mutants is significantly reduced upon environmental and genetic induction of fermentation. This anaerobic viability defect correlates with decreased membrane potential and is restored upon cation supplementation. Additionally, ypfP suppressor mutants exhibiting restored anaerobic viability harbor compensatory mutations in the LTA biosynthetic pathway that restore membrane potential. Overall, these results demonstrate that LTA maintains membrane potential during fermentative proliferation and promotes S. aureus metabolic versatility.

A Review of Cardiac Manifestations in Patients With Systemic Lupus Erythematosus and Antiphospholipid Syndrome With Focus on Endocarditis.

Patients with autoimmune diseases such as systemic lupus erythematosus (SLE) or antiphospholipid syndrome (APS) are at a higher risk for adverse cardiovascular events associated with increased morbidity and mortality. The increased risk of these events is often associated with rheumatic heart disease (heart valve or mural endocardium damage from rheumatic fever) following microbial infection (i.e., untreated or under-treated streptococcal infection). In particular, the weakening of cardiac vasculature due to rheumatic heart disease makes such patients with autoimmune diseases more susceptible to endocarditis. Endocarditis can be caused by an infection (infective endocarditis) or inflammation tied to disease activity (non-bacterial thrombotic endocarditis [NBTE]). Infective endocarditis among patients with autoimmune diseases may result from exposure to pathogens during dental or surgical procedures. NBTE commonly occurs as a result of fibrin and platelet aggregation on the cardiac valves without bacterial infection. While diagnosis and management can vary based on underlying etiology, an interdisciplinary approach that includes prevention and management from dentists, cardiologists, rheumatologists, and primary care physicians is needed. In addition, increasing patient and physician education on risk factors and prevention strategies is much needed. This manuscript will review the pathophysiology of endocarditis, the association between SLE and APS and endocarditis risk, the diagnosis and management of these autoimmune diseases with a focus on the prevention of cardiovascular disease risk, and make recommendations for diagnostic and management approaches to improve care.

Proceedings of the Ninth Annual Deep Brain Stimulation Think Tank: Advances in Cutting Edge Technologies, Artificial Intelligence, Neuromodulation, Neuroethics, Pain, Interventional Psychiatry, Epilepsy, and Traumatic Brain Injury.

DBS Think Tank IX was held on August 25-27, 2021 in Orlando FL with US based participants largely in person and overseas participants joining by video conferencing technology. The DBS Think Tank was founded in 2012 and provides an open platform where clinicians, engineers and researchers (from industry and academia) can freely discuss current and emerging deep brain stimulation (DBS) technologies as well as the logistical and ethical issues facing the field. The consensus among the DBS Think Tank IX speakers was that DBS expanded in its scope and has been applied to multiple brain disorders in an effort to modulate neural circuitry. After collectively sharing our experiences, it was estimated that globally more than 230,000 DBS devices have been implanted for neurological and neuropsychiatric disorders. As such, this year's meeting was focused on advances in the following areas: neuromodulation in Europe, Asia and Australia; cutting-edge technologies, neuroethics, interventional psychiatry, adaptive DBS, neuromodulation for pain, network neuromodulation for epilepsy and neuromodulation for traumatic brain injury.

Macrophage-Produced Peroxynitrite Induces Antibiotic Tolerance and Supersedes Intrinsic Mechanisms of Persister Formation.

Staphylococcus aureus is a leading human pathogen that frequently causes chronic and relapsing infections. Antibiotic-tolerant persister cells contribute to frequent antibiotic failure in patients. Macrophages represent an important niche during S. aureus bacteremia, and recent work has identified a role for oxidative burst in the formation of antibiotic-tolerant S. aureus. We find that host-derived peroxynitrite, the reaction product of superoxide and nitric oxide, is the main mediator of antibiotic tolerance in macrophages. Using a collection of S. aureus clinical isolates, we find that, despite significant variation in persister formation in pure culture, all strains were similarly enriched for antibiotic tolerance following internalization by activated macrophages. Our findings suggest that host interaction strongly induces antibiotic tolerance and may negate bacterial mechanisms of persister formation established in pure culture. These findings emphasize the importance of studying antibiotic tolerance in the context of bacterial interaction with the host and suggest that modulation of the host response may represent a viable therapeutic strategy to sensitize S. aureus to antibiotics.

DegP Initiates Regulated Processing of Filamentous Hemagglutinin in Bordetella bronchiseptica.

Filamentous hemagglutinin (FhaB) is a critical virulence factor for both Bordetella pertussis, the causal agent of whooping cough, and the closely related species Bordetella bronchiseptica. FhaB is an adhesin, suppresses inflammatory cytokine production, and protects against phagocytic cell clearance during infection. Regulated degradation of the FhaB C-terminal prodomain is required to establish a persistent infection in mice. Two proteases, CtpA in the periplasm and SphB1 on the bacterial surface, are known to mediate FhaB processing, and we recently determined that CtpA functions before, and controls the FhaB cleavage site of, SphB1. However, the data indicate that another periplasmic protease must initiate degradation of the prodomain by removing a portion of the FhaB C terminus that inhibits CtpA-mediated degradation. Using a candidate approach, we identified DegP as the initiating protease. Deletion of degP or substitution of its predicted catalytic residue resulted in reduced creation of FHA' (the main product of FhaB processing) and an accumulation of full-length FhaB in whole-cell lysates. Also, FHA' was no longer released into culture supernatants in degP mutants. Alterations of the FhaB C terminus that relieve inhibition of CtpA abrogate the need for DegP, consistent with DegP functioning prior to CtpA in the processing pathway. DegP is not required for secretion of FhaB through FhaC or for adherence of the bacteria to host cells, indicating that DegP acts primarily as a protease and not a chaperone for FhaB in B. bronchiseptica. Our results highlight a role for HtrA family proteases in activation of virulence factors in pathogenic bacteria. IMPORTANCE Two-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and we here identified the protease that initiates a processing cascade that controls FhaB function. Our results are significant because they provide insight into the molecular mechanism underlying the ability of Bordetella species to prevent clearance by phagocytic cells, which is critical for bacterial persistence in the lower respiratory tract. Our findings also highlight an underappreciated role for HtrA family proteases in processing specific bacterial virulence factors.

The Staphylococcus aureus Cystine Transporters TcyABC and TcyP Facilitate Nutrient Sulfur Acquisition during Infection.

Staphylococcus aureus is a significant human pathogen due to its capacity to cause a multitude of diseases. As such, S. aureus efficiently pillages vital nutrients from the host; however, the molecular mechanisms that support sulfur acquisition during infection have not been established. One of the most abundant extracellular sulfur-containing metabolites within the host is cysteine, which acts as the major redox buffer in the blood by transitioning between reduced and oxidized (cystine) forms. We therefore hypothesized that S. aureus acquires host-derived cysteine and cystine as sources of nutrient sulfur during systemic infection. To test this hypothesis, we used the toxic cystine analogue selenocystine to initially characterize S. aureus homologues of the Bacillus subtilis cystine transporters TcyABC and TcyP. We found that genetic inactivation of both TcyA and TcyP induced selenocystine resistance. The double mutant also failed to proliferate in medium supplemented with cystine, cysteine, or N-acetyl cysteine as the sole sulfur source. However, only TcyABC was necessary for proliferation in defined medium containing homocystine as the sulfur source. Using a murine model of systemic infection, we observed tcyP-dependent competitive defects in the liver and heart, indicating that this sulfur acquisition strategy supports proliferation of S. aureus in these organs. Phylogenetic analyses identified TcyP homologues in many pathogenic species, implying that this sulfur procurement strategy is conserved. In total, this study is the first to experimentally validate sulfur acquisition systems in S. aureus and establish their importance during pathogenesis.

Resurrection of antibiotics that methicillin-resistant Staphylococcus aureus resists by silver-doped bioactive glass-ceramic microparticles.

This work describes a novel strategy to combat methicillin-resistant Staphylococcus aureus (MRSA) via the reactivation of inert antibiotics. This strategy exploits a multifunctional system consisting of bioactive glass-ceramic microparticles with antibacterial properties combined with various antibiotics to kill MRSA. Specifically, sol-gel derived silver-doped bioactive glass-ceramic microparticles (Ag-BG) combined with antibiotics that MRSA resists such as oxacillin or fosfomycin, significantly decreased the viability of MRSA. Ag-BG also potentiated the activity of vancomycin on static bacteria, which are typically resistant to this antibiotic. Notably, the synergistic activity is restricted to cell-envelope acting antibiotics as Ag-BG supplementation did not increase the efficacy of gentamicin. Bacteria viability assays and electron microscopy images demonstrate that Ag-BG synergizes to restore antibacterial activity to antibiotics that MRSA resists. The low cytotoxicity previously studied against oral bacteria, together with the known regenerative properties presented in previous studies, and the unique antibacterial properties observed in this work when they are combined with antibiotics, make this multifunctional system a promising approach for healing infected tissue. STATEMENT OF SIGNIFICANCE: This study addresses a very significant issue in the field of antibiotic resistance presenting an innovative way to clear MRSA, by utilizing bioactive glass-ceramic microparticles in combination with antibiotics. Multifunctional glass-ceramic microparticles doped with silver ions (Ag-BG) have been previously observed to exhibit bioactive and antibacterial properties. In this study Ag-BG microparticles were observed to synergize with antibiotics restoring their sensitivity against MRSA. This research work presents a novel approach to resurrect ineffective antibiotics and render them effective against MRSA. Cytotoxicity to eukaryotic cells is not anticipated, as it has been previously observed that these microparticles can trigger hard and soft dental tissue regeneration, when they are utilized in certain concentrations. This study opens a new avenue in the treatment of multidrug resistance bacteria.

Metabolic Syndrome Induces Release of Smaller Extracellular Vesicles from Porcine Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) belong to the endogenous cellular reparative system, and can be used exogenously in cell-based therapy. MSCs release extracellular vesicles (EVs), including exosomes and microvesicles, which mediate some of their therapeutic activity through intercellular communication. We have previously demonstrated that metabolic syndrome (MetS) modifies the cargo packed within swine EV, but whether it influences their phenotypical characteristics remains unclear. This study tested the hypothesis that MetS shifts the size distribution of MSC-derived EVs. Adipose tissue-derived MSC-EV subpopulations from Lean (n = 6) and MetS (n = 6) pigs were characterized for number and size using nanoparticle-tracking analysis, flow cytometry, and transmission electron microscopy. Expression of exosomal genes was determined using next-generation RNA-sequencing (RNA-seq). The number of EV released from Lean and MetS pig MSCs was similar, yet MetS-MSCs yielded a higher proportion of small-size EVs (202.4 ± 17.7 nm vs. 280.3 ± 15.1 nm), consistent with exosomes. RNA-seq showed that their EVs were enriched with exosomal markers. Lysosomal activity remained unaltered in MetS-MSCs. Therefore, MetS alters the size distribution of MSC-derived EVs in favor of exosome release. These observations may reflect MSC injury and membrane recycling in MetS or increased expulsion of waste products, and may have important implications for development of adequate cell-based treatments.

Staphylococcus aureus Utilizes Host-Derived Lipoprotein Particles as Sources of Fatty Acids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health. Consequently, much effort has focused on the development of new antimicrobials that target novel aspects of S. aureus physiology. Fatty acids are required to maintain cell viability, and bacteria synthesize fatty acids using the type II fatty acid synthesis (FASII) pathway. FASII is significantly different from human fatty acid synthesis, underscoring the therapeutic potential of inhibiting this pathway. However, many Gram-positive pathogens incorporate exogenous fatty acids, bypassing FASII inhibition and leaving the clinical potential of FASII inhibitors uncertain. Importantly, the source(s) of fatty acids available to pathogens within the host environment remains unclear. Fatty acids are transported throughout the body by lipoprotein particles in the form of triglycerides and esterified cholesterol. Thus, lipoproteins, such as low-density lipoprotein (LDL), represent a potentially rich source of exogenous fatty acids for S. aureus during infection. We sought to test the ability of LDLs to serve as a fatty acid source for S. aureus and show that cells cultured in the presence of human LDLs demonstrate increased tolerance to the FASII inhibitor triclosan. Using mass spectrometry, we observed that host-derived fatty acids present in the LDLs are incorporated into the staphylococcal membrane and that tolerance to triclosan is facilitated by the fatty acid kinase A, FakA, and Geh, a triacylglycerol lipase. Finally, we demonstrate that human LDLs support the growth of S. aureus fatty acid auxotrophs. Together, these results suggest that human lipoprotein particles are a viable source of exogenous fatty acids for S. aureus during infection.IMPORTANCE Inhibition of bacterial fatty acid synthesis is a promising approach to combating infections caused by S. aureus and other human pathogens. However, S. aureus incorporates exogenous fatty acids into its phospholipid bilayer. Therefore, the clinical utility of targeting bacterial fatty acid synthesis is debated. Moreover, the fatty acid reservoir(s) exploited by S. aureus is not well understood. Human low-density lipoprotein particles represent a particularly abundant in vivo source of fatty acids and are present in tissues that S. aureus colonizes. Herein, we establish that S. aureus is capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis. These findings imply that S. aureus targets LDLs as a source of fatty acids during pathogenesis.

Moral Mentation: What Neurocognitive Studies of Psychopathy May Really Offer the Internalism/Externalism Debate.

A persistent debate about moral capacity - and neuroethics - focuses upon the internalism-externalism controversy. Internalism holds that moral judgments necessarily motivate an agent's actions; externalism views moral judgments as not inherently motivating an agent to perform moral actions. Neuroethical discussions of the putative cognitive basis of moral thought and action would be better informed if neurocognitive research would yield data sufficient for validating one side or the other. Neuroscientific studies of psychopaths have been employed in this regard. However, it seems that neuroscientific investigations to date have been inadequate to wholly define the nature of moral knowledge, and thus fail to preferentially support (or foster) an exclusively internalist or externalist view. Thus, moving forward it will be necessary to carefully define questions that neuroscience is employed to address and answer, and to ensure that empirical findings are not distorted to support preconceived theoretical assumptions. In this way, neuroscientific investigations can be used in a conciliatory way to both balance views of processes operative in moral cognition, and raise ethical, legal, and social questions about what research findings actually mean, and what medicine - and societies - will do with such information and meanings.

A four-part working bibliography of neuroethics: Part 4 - Ethical issues in clinical and social applications of neuroscience.

BACKGROUND: As a discipline, neuroethics addresses a range of questions and issues generated by basic neuroscientific research (inclusive of studies of putative neurobiological processes involved in moral and ethical cognition and behavior), and its use and meanings in the clinical and social spheres. Here, we present Part 4 of a four-part bibliography of the neuroethics literature focusing on clinical and social applications of neuroscience, to include: the treatment-enhancement discourse; issues arising in neurology, psychiatry, and pain care; neuroethics education and training; neuroethics and the law; neuroethics and policy and political issues; international neuroethics; and discourses addressing "trans-" and "post-" humanity. METHODS: To complete a systematic survey of the literature, 19 databases and 4 individual open-access journals were employed. Searches were conducted using the indexing language of the U.S. National Library of Medicine (NLM). A Python code was used to eliminate duplications in the final bibliography. RESULTS: When taken with Parts 1-3, this bibliography aims to provide a listing of international peerreviewed papers, books, and book chapters published from 2002 through 2016. While seeking to be as comprehensive as possible, it may be that some works were inadvertently and unintentionally not included. We therefore invite commentary from the field to afford completeness and contribute to this bibliography as a participatory work-in-progress.

Neuroethics beyond Normal.

An integrated and principled neuroethics offers ethical guidelines able to transcend conventional and medical reliance on normality standards. Elsewhere we have proposed four principles for wise guidance on human transformations. Principles like these are already urgently needed, as bio- and cyberenhancements are rapidly emerging. Context matters. Neither "treatments" nor "enhancements" are objectively identifiable apart from performance expectations, social contexts, and civic orders. Lessons learned from disability studies about enablement and inclusion suggest a fresh way to categorize modifications to the body and its performance. The term "enhancement" should be broken apart to permit recognition of enablements and augmentations, and kinds of radical augmentation for specialized performance. Augmentations affecting the self, self-worth, and self-identity of persons require heightened ethical scrutiny. Reversibility becomes the core problem, not the easy answer, as augmented persons may not cooperate with either decommissioning or displacement into unaccommodating societies. We conclude by indicating how our four principles of self-creativity, nonobsolescence, empowerment, and citizenship establish a neuroethics beyond normal that is better prepared for a future in which humans and their societies are going so far beyond normal.