Updates on Topical Dyad and Triple Combination Therapies Approved for Acne Vulgaris.

Acne vulgaris is a multifaceted disease characterized by inflammatory and noninflammatory lesions. Topical combination therapies offer a multifaceted approach to acne treatment, with synergistic effects and a broad spectrum of action against multiple factors in acne pathogenesis in one single formulation. Clindamycin phosphate/benzoyl peroxide/adapalene, a combination therapy consisting of clindamycin phosphate 1.2%, benzoyl peroxide (BPO) 3.1%, and adapalene 0.15%, is a novel treatment, the only FDA-approved triple combination drug that offers effective treatment of acne vulgaris. This review aims to provide information on clindamycin phosphate/benzoyl peroxide/adapalene and review the literature on combination topical acne medications approved in the United States. This search was conducted on topical combination therapies for acne, their efficacy, adverse effects, and impacts on quality of life with a specific focus on the newly approved clindamycin phosphate/benzoyl peroxide/adapalene and its sub-component dyads, along with other combinations. PubMed, SCOPUS, Embase, Cochrane, and Web of Science databases were searched for publications in 2018-2023. Primary sources were given priority, and secondary sources such as other reviews were considered to supplement any missing information. It was found that various topical dyad and triad combinations exist for acne vulgaris, including adapalene/BPO, tazarotene/clindamycin, clindamycin/BPO, adapalene/clindamycin, topical tretinoin/azelaic acid, topical tretinoin/BPO, and clindamycin phosphate/benzoyl peroxide/adapalene. Dyad and triple combinations represent a promising, convenient solution for acne management, potentially improving patient adherence due to its single formulation. Clindamycin phosphate/benzoyl peroxide/adapalene exhibited significantly high efficacy in treating both inflammatory and noninflammatory lesions, a minimal side effect profile, although no significant changes in quality-of-life measures. Further research is indicated to assess its long-term efficacy and impact on other acne metrics such as cost, scarring, psychosocial implications, and impact on diverse patient populations.

Infection induced inflammation impairs wound healing through IL-1ß signaling.

Wound healing is impaired by infection; however, how microbe-induced inflammation modulates tissue repair remains unclear. We took advantage of the optical transparency of zebrafish and a genetically tractable microbe, Listeria monocytogenes, to probe the role of infection and inflammation in wound healing. Infection with bacteria engineered to activate the inflammasome, Lm-Pyro, induced persistent inflammation and impaired healing despite low bacterial burden. Inflammatory infections induced il1b expression and blocking IL-1R signaling partially rescued wound healing in the presence of persistent infection. We found a critical window of microbial clearance necessary to limit persistent inflammation and enable efficient wound repair. Taken together, our findings suggest that the dynamics of microbe-induced tissue inflammation impacts repair in complex tissue damage independent of bacterial load, with a critical early window for efficient tissue repair.

Assessing Longitudinal Treatment Efficacies and Alterations in Molecular Markers Associated with Glutamatergic Signaling and Immune Checkpoint Inhibitors in a Spontaneous Melanoma Mouse Model.

Previous work done by our laboratory described the use of an immunocompetent spontaneous melanoma-prone mouse model, TGS (TG-3/SKH-1), to evaluate treatment outcomes using inhibitors of glutamatergic signaling and immune checkpoint for 18 weeks. We showed a significant therapeutic efficacy with a notable sex-biased response in male mice. In this follow-up 18-week study, the dose of the glutamatergic signaling inhibitor was increased (from 1.7 mg/kg to 25 mg/kg), which resulted in improved responses in female mice but not male mice. The greatest reduction in tumor progression was observed in male mice treated with single-agent troriluzole and anti-PD-1. Furthermore, a randomly selected group of mice was removed from treatment after 18 weeks and maintained for up to an additional 48 weeks demonstrating the utility of the TGS mouse model to perform a ≥1-year preclinical therapeutic study in a physiologically relevant tumor-host environment. Digital spatial imaging analyses were performed in tumors and tumor microenvironments across treatment modalities using antibody panels for immune cell types and immune cell activation. The results suggest that immune cell populations and cytotoxic activities of T cells play critical roles in treatment responses in these mice. Examination of a group of molecular protein markers based on the proposed mechanisms of action of inhibitors of glutamatergic signaling and immune checkpoint showed that alterations in expression levels of xCT, γ-H2AX, EAAT2, PD-L1, and PD-1 are likely associated with the loss of treatment responses. These results suggest the importance of tracking changes in molecular markers associated with the mechanism of action of therapeutics over the course of a longitudinal preclinical therapeutic study in spatial and temporal manners.

C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression in Listeria monocytogenes.

C-di-AMP is an essential second messenger in many bacteria but its levels must be regulated. Unregulated c-di-AMP accumulation attenuates the virulence of many bacterial pathogens, including those that do not require c-di-AMP for growth. However, the mechanisms by which c-di-AMP regulates bacterial pathogenesis remain poorly understood. In Listeria monocytogenes , a mutant lacking both c-di-AMP phosphodiesterases, denoted as the ΔPDE mutant, accumulates a high c-di-AMP level and is significantly attenuated in the mouse model of systemic infection. All key L. monocytogenes virulence genes are transcriptionally upregulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Our transcriptomic analysis revealed that the ΔPDE mutant is significantly impaired for the expression of virulence genes within the PrfA core regulon. Subsequent quantitative gene expression analyses validated this phenotype both at the basal level and upon PrfA activation by GSH. A constitutively active PrfA * variant, PrfA G145S, which mimics the GSH-bound conformation, restores virulence gene expression in ΔPDE but only partially rescues virulence defect. Through GSH quantification and uptake assays, we found that the ΔPDE strain is significantly depleted for GSH, and that c-di-AMP inhibits GSH uptake. Constitutive expression of gshF (encoding a GSH synthetase) does not restore GSH levels in the ΔPDE strain, suggesting that c-di-AMP inhibits GSH synthesis activity or promotes GSH catabolism. Taken together, our data reveals GSH metabolism as another pathway that is regulated by c-di-AMP. C-di-AMP accumulation depletes cytoplasmic GSH levels within L. monocytogenes that leads to impaired virulence program expression. IMPORTANCE: C-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy, and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the mechanisms by which c-di-AMP accumulation diminishes bacterial pathogenesis are poorly understood. Such understanding will reveal the molecular function of c-di-AMP, and inform therapeutic development strategies. Here, we identify GSH metabolism as a pathway regulated by c-di-AMP that is pertinent to L. monocytogenes replication in the host. Given the role of GSH as a virulence signal, nutrient, and antioxidant, GSH depletion impairs virulence program expression and likely diminishes host adaptation.

Maintenance of heme homeostasis in Staphylococcus aureus through post-translational regulation of glutamyl-tRNA reductase.

Staphylococcus aureus is an important human pathogen responsible for a variety of infections including skin and soft tissue infections, endocarditis, and sepsis. The combination of increasing antibiotic resistance in this pathogen and the lack of an efficacious vaccine underscores the importance of understanding how S. aureus maintains metabolic homeostasis in a variety of environments, particularly during infection. Within the host, S. aureus must regulate cellular levels of the cofactor heme to support enzymatic activities without encountering heme toxicity. Glutamyl tRNA reductase (GtrR), the enzyme catalyzing the first committed step in heme synthesis, is an important regulatory node of heme synthesis in Bacteria, Archaea, and Plantae. In many organisms, heme status negatively regulates the abundance of GtrR, controlling flux through the heme synthesis pathway. We identified two residues within GtrR, H32 and R214, that are important for GtrR-heme binding. However, in strains expressing either GtrRH32A or GtrRR214A, heme homeostasis was not perturbed, suggesting an alternative mechanism of heme synthesis regulation occurs in S. aureus. In this regard, we report that heme synthesis is regulated through phosphorylation and dephosphorylation of GtrR by the serine/threonine kinase Stk1 and the phosphatase Stp1, respectively. Taken together, these results suggest that the mechanisms governing staphylococcal heme synthesis integrate both the availability of heme and the growth status of the cell. IMPORTANCE Staphylococcus aureus represents a significant threat to human health. Heme is an iron-containing enzymatic cofactor that can be toxic at elevated levels. During infection, S. aureus must control heme levels to replicate and survive within the hostile host environment. We identified residues within a heme biosynthetic enzyme that are critical for heme binding in vitro; however, abrogation of heme binding is not sufficient to perturb heme homeostasis within S. aureus. This marks a divergence from previously reported mechanisms of heme-dependent regulation of the highly conserved enzyme glutamyl tRNA reductase (GtrR). Additionally, we link cell growth arrest to the modulation of heme levels through the post-translational regulation of GtrR by the kinase Stk1 and the phosphatase Stp1.

Characterization of an Autoinducing Peptide Signal Reveals Highly Efficacious Synthetic Inhibitors and Activators of Quorum Sensing and Biofilm Formation in Listeria monocytogenes.

Bacteria can use chemical signals to assess their local population density in a process called quorum sensing (QS). Many of these bacteria are common pathogens, including Gram-positive bacteria that utilize agr QS systems regulated by macrocyclic autoinducing peptide (AIP) signals. Listeria monocytogenes, an important foodborne pathogen, uses an agr system to regulate a variety of virulence factors and biofilm formation, yet little is known about the specific roles of agr in Listeria infection and its persistence in various environments. Herein, we report synthetic peptide tools that will enable the study of QS in Listeria. We identified a 6-mer AIP signal in L. monocytogenes supernatants and selected it as a scaffold around which a collection of non-native AIP mimics was designed and synthesized. These peptides were evaluated in cell-based agr reporter assays to generate structure-activity relationships for AIP-based agonism and antagonism in L. monocytogenes. We discovered synthetic agonists with increased potency relative to native AIP and a synthetic antagonist capable of reducing agr activity to basal levels. Notably, the latter peptide was able to reduce biofilm formation by over 90%, a first for a synthetic QS modulator in wild-type L. monocytogenes. The lead agr agonist and antagonist in L. monocytogenes were also capable of antagonizing agr signaling in the related pathogen Staphylococcus aureus, further extending their utility and suggesting different mechanisms of agr activation in these two pathogens. This study represents an important first step in the application of chemical methods to modulate QS and concomitant virulence outcomes in L. monocytogenes.

Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence.

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD+ while also generating a proton motive force. The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respirations. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD+/NADH ratio through expression of water-forming NADH oxidase could rescue phenotypes associated with DHNA deficiency. Here, we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes. Furthermore, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of its role in the extracellular electron transport pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.

Type I Interferon Signaling on Antigen-Presenting Cells Blunts Cell-Mediated Immunity toward Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen that has been used for decades to understand mechanisms of bacterial pathogenesis and both innate and adaptive immunity. L. monocytogenes is a potent activator of CD8+ T-cell-mediated immunity, yet how the innate immune response to infection modulates CD8+ T-cell responses is incompletely understood. Here, we address how two innate immune pathways triggered by L. monocytogenes, type I interferon (IFN) production and inflammasome activation, impact the CD8+ T-cell response. We utilized a combination of mutant mice and genetically engineered L. monocytogenes to address this question. Mice lacking the type I IFN receptor (IFNAR-/-) had the most robust T-cell response, while caspase-1-/- mice were not different from wild type (WT). Caspase-1-/-/IFNAR-/- mice had fewer T-cells than IFNAR-/- mice, suggesting a role for inflammasome activation in the absence of type I IFN. IFNAR-/- had more than twice as many memory precursors promoting enhanced protection from rechallenge. Importantly, short-lived effectors were equivalent in all strains of mice. L. monocytogenes strains genetically modified to induce lower type I interferon production yielded enhanced T-cell responses. IFNAR-/- dendritic cells induced more T-cells to proliferate than WT in ex vivo T-cell proliferation assays, suggesting deficits from type I interferon signaling may be dendritic cell intrinsic, rather than acting on T-cells. Thus, modulating type I IFN signaling during vaccination may lead to more potent T-cell-based vaccines. Importantly, this suggests innate immune signaling significantly impacts the CD8+ T-cell response and suggests CD8+ T-cell quantity and quality are important factors to consider during rational vaccine design.

A tessellated lymphoid network provides whole-body T cell surveillance in zebrafish.

Homeostatic trafficking to lymph nodes allows T cells to efficiently survey the host for cognate antigen. Nonmammalian jawed vertebrates lack lymph nodes but maintain diverse T cell pools. Here, we exploit in vivo imaging of transparent zebrafish to investigate how T cells organize and survey for antigen in an animal devoid of lymph nodes. We find that naïve-like T cells in zebrafish organize into a previously undescribed whole-body lymphoid network that supports streaming migration and coordinated trafficking through the host. This network has the cellular hallmarks of a mammalian lymph node, including naïve T cells and CCR7-ligand expressing nonhematopoietic cells, and facilitates rapid collective migration. During infection, T cells transition to a random walk that supports antigen-presenting cell interactions and subsequent activation. Our results reveal that T cells can toggle between collective migration and individual random walks to prioritize either large-scale trafficking or antigen search in situ. This lymphoid network thus facilitates whole-body T cell trafficking and antigen surveillance in the absence of a lymph node system.

Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence.

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions against these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell-wall stress, cytosolic survival, inflammasome avoidance, and, ultimately, virulence in vivo. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggests that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell-wall stress responses and virulence in vivo. Taken together, these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes. IMPORTANCE Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol. GlmR is important for resistance to some cell-wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell-wall metabolite, UDP-GlcNAc, and suppressors that increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc, and this enzymatic activity is conserved in both Bacillus subtilis and Staphylococcus aureus. These results highlight the importance of accessory cell wall metabolism enzymes in responding to cell-wall stress in a variety of Gram-positive bacteria.

A tessellated lymphoid network provides whole-body T cell surveillance in zebrafish.

Homeostatic trafficking to lymph nodes allows T cells to efficiently survey the host for cognate antigen. Non-mammalian jawed vertebrates lack lymph nodes but maintain similarly diverse T cell pools. Here, we exploit in vivo imaging of transparent zebrafish to investigate how T cells organize and survey for antigen in an animal devoid of lymph nodes. We find that naïve-like T cells in zebrafish organize into a previously undescribed whole-body lymphoid network that supports streaming migration and coordinated trafficking through the host. This network has the cellular hallmarks of a mammalian lymph node, including naïve T cells and CCR7-ligand expressing non-hematopoietic cells, and facilitates rapid collective migration. During infection, T cells transition to a random walk that supports antigen presenting cell interactions and subsequent activation. Our results reveal that T cells can toggle between collective migration and individual random walks to prioritize either large-scale trafficking or antigen search in situ . This novel lymphoid network thus facilitates whole-body T cell trafficking and antigen surveillance in the absence of a lymph node system. Significance Statement: In mammals, lymph nodes play a critical role in the initiation of adaptive immune responses by providing a dedicated place for T cells to scan antigen-presenting cells. Birds, reptiles, amphibians, and fish all maintain diverse repertoires of T cells but lack lymph nodes, raising questions about how adaptive immunity functions in lower jawed vertebrates. Here, we describe a novel network of lymphocytes in zebrafish that supports whole-body T cell trafficking and provides a site for antigen search, mirroring the function of mammalian lymph nodes. Within this network, T cells can prioritize large-scale trafficking or antigen scanning by toggling between two distinct modes of migration. This network provides valuable insights into the evolution of adaptive immunity.

Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence.

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD + while also generating a proton motive force (PMF). The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respiration. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD + /NADH ratio through expression of water-forming NADH oxidase (NOX) could rescue phenotypes associated with DHNA deficiency. Here we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes . Further, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of the extracellular electron transport (EET) pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.

Modeled distribution shifts of North American birds over four decades based on suitable climate alone do not predict observed shifts.

As climate change alters the global environment, it is critical to understand the relationship between shifting climate suitability and species distributions. Key questions include whether observed changes in population abundance are aligned with the velocity and direction of shifts predicted by climate suitability models and if the responses are consistent among species with similar ecological traits. We examined the direction and velocity of the observed abundance-based distribution centroids compared with the model-predicted bioclimatic distribution centroids of 250 bird species across the United States from 1969 to 2011. We hypothesized that there is a significant positive correlation in both direction and velocity between the observed and the modeled shifts. We then tested five additional hypotheses that predicted differential shifting velocity based on ecological adaptability and climate change exposure. Contrary to our hypotheses, we found large differences between the observed and modeled shifts among all studied bird species and within specific ecological guilds. However, temperate migrants and habitat generalist species tended to have higher velocity of observed shifts than other species. Neotropical migratory and wetland birds also had significantly different observed velocities than their counterparts, which may be due to their climate change exposure. The velocity based on modeled bioclimatic suitability did not exhibit significant differences among most guilds. Boreal forest birds were the only guild with significantly faster modeled-shifts than the other groups, suggesting an elevated conservation risk for high latitude and altitude species. The highly idiosyncratic species responses to climate and the mismatch between shifts in modeled and observed distribution centroids highlight the challenge of predicting species distribution change based solely on climate suitability and the importance of non-climatic factors traits in shaping species distributions.

NAD(H)-loaded nanoparticles for efficient sepsis therapy via modulating immune and vascular homeostasis.

Sepsis is a life-threatening organ dysfunction responsible for nearly 270,000 deaths annually in the United States alone. Nicotinamide adenine dinucleotide (NAD+), an immunomodulator, can potentially treat sepsis; however, clinical application of NAD+ is hindered by its inability to be directly taken up by cells. To address this challenge, a family of nanoparticles (NPs) loaded with either NAD+ or the reduced form of NAD+ (NADH), hereafter NAD(H)-loaded NPs, were engineered to enable direct cellular transport and replenishment of NAD(H). The NAD(H)-loaded NPs improved cellular energy supply, suppressed inflammation and prevented inflammation-induced cell pyroptosis and apoptosis. Therefore, the NPs can help maintain immune homoeostasis and vascular function, two key factors in the pathogenesis of sepsis. The NAD(H)-loaded NPs demonstrated excellent therapeutic efficacies in treating endotoxemia and multidrug-resistant pathogen-induced bacteremia. In addition, the NAD(H)-loaded NPs prevented caecal ligation and puncture-induced multiorgan injury and improved outcomes of secondary Pseudomonas aeruginosa infections following caecal ligation and puncture, thus potentially leading to a highly innovative and translational approach to treat sepsis efficiently and safely.

eTRANSAFE: Building a sustainable framework to share reproducible drug safety knowledge with the public domain.

Integrative drug safety research in translational health informatics has rapidly evolved and included data that are drawn in from many resources, combining diverse data that are either reused from (curated) repositories, or newly generated at source. Each resource is mandated by different sets of metadata rules that are imposed on the incoming data. Combination of the data cannot be readily achieved without interference of data stewardship and the top-down policy guidelines that supervise and inform the process for data combination to aid meaningful interpretation and analysis of such data. The eTRANSAFE Consortium's effort to drive integrative drug safety research at a large scale hereby present the lessons learnt and the proposal of solution at the guidelines in practice at this Innovative Medicines Initiative (IMI) project. Recommendations in these guidelines were compiled from feedback received from key stakeholders in regulatory agencies, EFPIA companies, and academic partners. The research reproducibility guidelines presented in this study lay the foundation for a comprehensive data sharing and knowledge management plans accounting for research data management in the drug safety space - FAIR data sharing guidelines, and the model verification guidelines as generic deliverables that best practices that can be reused by other scientific community members at large. FAIR data sharing is a dynamic landscape that rapidly evolves with fast-paced technology advancements. The research reproducibility in drug safety guidelines introduced in this study provides a reusable framework that can be adopted by other research communities that aim to integrate public and private data in biomedical research space.

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Cellular respiration is one of the main ways organisms make energy. It works by linking the oxidation of an electron donor (like sugar) to the reduction of an electron acceptor (like oxygen). Electrons pass between the two molecules along what is known as an 'electron transport chain'. This process generates a force that powers the production of adenosine triphosphate (ATP), a molecule that cells use to store energy. Respiration is a common way for cells to replenish their energy stores, but it is not the only way. A simpler process that does not require a separate electron acceptor or an electron transport chain is called fermentation. Many bacteria have the capacity to perform both respiration and fermentation and do so in a context-dependent manner. Research has shown that respiration can contribute to bacterial diseases, like tuberculosis and listeriosis (a disease caused by the foodborne pathogen Listeria monocytogenes). Indeed, some antibiotics even target bacterial respiration. Despite being often discussed in the context of generating ATP, respiration is also important for many other cellular processes, including maintaining the balance of reduced and oxidized nicotinamide adenine dinucleotide (NAD) cofactors. Because of these multiple functions, the exact role respiration plays in disease is unknown. To find out more, Rivera-Lugo, Deng et al. developed strains of the bacterial pathogen Listeria monocytogenes that lacked some of the genes used in respiration. The resulting bacteria were still able to produce energy, but they became much worse at infecting mammalian cells. The use of a genetic tool that restored the balance of reduced and oxidized NAD cofactors revived the ability of respiration-deficient L. monocytogenes to infect mammalian cells, indicating that this balance is what the bacterium requires to infect. Research into respiration tends to focus on its role in generating ATP. But these results show that for some bacteria, this might not be the most important part of the process. Understanding the other roles of respiration could change the way that researchers develop antibacterial drugs in the future. This in turn could help with the growing problem of antibiotic resistance.

In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish.

The function of macrophages in vitro is linked to their metabolic rewiring. However, macrophage metabolism remains poorly characterized in situ. Here, we used two-photon intensity and lifetime imaging of autofluorescent metabolic coenzymes, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), to assess the metabolism of macrophages in the wound microenvironment. Inhibiting glycolysis reduced NAD(P)H mean lifetime and made the intracellular redox state of macrophages more oxidized, as indicated by reduced optical redox ratio. We found that TNFα+ macrophages had lower NAD(P)H mean lifetime and were more oxidized compared to TNFα- macrophages. Both infection and thermal injury induced a macrophage population with a more oxidized redox state in wounded tissues. Kinetic analysis detected temporal changes in the optical redox ratio during tissue repair, revealing a shift toward a more reduced redox state over time. Metformin reduced TNFα+ wound macrophages, made intracellular redox state more reduced and improved tissue repair. By contrast, depletion of STAT6 increased TNFα+ wound macrophages, made redox state more oxidized and impaired regeneration. Our findings suggest that autofluorescence of NAD(P)H and FAD is sensitive to dynamic changes in intracellular metabolism in tissues and can be used to probe the temporal and spatial regulation of macrophage metabolism during tissue damage and repair.