1,25 dihydroxyvitamin D3-mediated effects on bovine innate immunity and on biofilm-forming Staphylococcus spp. isolated from cattle with mastitis.

Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host's immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0-200 nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72 h. Concentrations of 0-100 mM for 24 h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24 h decreased the internalization of S. aureus V329 into MAC-T cells (0-100 nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0-10 nM). Calcitriol and two conditioned media, obtained by treating the cells with 25-200 nM of the metabolite for 24 h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0-200 nM for 12 and 24 h, respectively). In contrast, the conditioned media (0-100 nM for 24 h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host's endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol's potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.

CFTR is required for zinc-mediated antibacterial defense in human macrophages.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion transporter required for epithelial homeostasis in the lung and other organs, with CFTR mutations leading to the autosomal recessive genetic disease CF. Apart from excessive mucus accumulation and dysregulated inflammation in the airways, people with CF (pwCF) exhibit defective innate immune responses and are susceptible to bacterial respiratory pathogens such as Pseudomonas aeruginosa. Here, we investigated the role of CFTR in macrophage antimicrobial responses, including the zinc toxicity response that is used by these innate immune cells against intracellular bacteria. Using both pharmacological approaches, as well as cells derived from pwCF, we show that CFTR is required for uptake and clearance of pathogenic Escherichia coli by CSF-1-derived primary human macrophages. CFTR was also required for E. coli-induced zinc accumulation and zinc vesicle formation in these cells, and E. coli residing in macrophages exhibited reduced zinc stress in the absence of CFTR function. Accordingly, CFTR was essential for reducing the intramacrophage survival of a zinc-sensitive E. coli mutant compared to wild-type E. coli. Ectopic expression of the zinc transporter SLC30A1 or treatment with exogenous zinc was sufficient to restore antimicrobial responses against E. coli in human macrophages. Zinc supplementation also restored bacterial killing in GM-CSF-derived primary human macrophages responding to P. aeruginosa, used as an in vitro macrophage model relevant to CF. Thus, restoration of the zinc toxicity response could be pursued as a therapeutic strategy to restore innate immune function and effective host defense in pwCF.

Histoplasma capsulatum Relies on Tryptophan Biosynthesis To Proliferate within the Macrophage Phagosome.

Histoplasma capsulatum yeasts reside and proliferate within the macrophage phagosome during infection. This nutrient-depleted phagosomal environment imposes challenges to Histoplasma yeasts for nutrition acquisition. Histoplasma yeasts require all 20 amino acids, which can be formed by de novo biosynthesis and/or acquired directly from the phagosomal environment. We investigated how Histoplasma obtains aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) within the phagosome during infection of macrophages. Depletion of key enzymes of the phenylalanine or tyrosine biosynthetic pathway neither impaired Histoplasma's ability to proliferate within macrophages nor resulted in attenuated virulence in vivo. However, loss of tryptophan biosynthesis resulted in reduced growth within macrophages and severely attenuated virulence in vivo. Together, these results indicate that phenylalanine and tyrosine, but not tryptophan, are available to Histoplasma within the macrophage phagosome. The herbicide glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase of the aromatic amino acid biosynthetic pathway, inhibited Histoplasma yeast growth, and this growth inhibition was partially reversed by aromatic amino acid supplementation or overexpression of ARO1. These results suggest that the aromatic amino acid biosynthetic pathway is a candidate drug target to develop novel antifungal therapeutics.

CRISPR Interference Reveals That All-Trans-Retinoic Acid Promotes Macrophage Control of Mycobacterium tuberculosis by Limiting Bacterial Access to Cholesterol and Propionyl Coenzyme A.

Macrophages are a protective replicative niche for Mycobacterium tuberculosis (Mtb) but can kill the infecting bacterium when appropriately activated. To identify mechanisms of clearance, we compared levels of bacterial restriction by human macrophages after treatment with 26 compounds, including some currently in clinical trials for tuberculosis. All-trans-retinoic acid (ATRA), an active metabolite of vitamin A, drove the greatest increase in Mtb control. Bacterial clearance was transcriptionally and functionally associated with changes in macrophage cholesterol trafficking and lipid metabolism. To determine how these macrophage changes affected bacterial control, we performed the first Mtb CRISPR interference screen in an infection model, identifying Mtb genes specifically required to survive in ATRA-activated macrophages. These data showed that ATRA treatment starves Mtb of cholesterol and the downstream metabolite propionyl coenzyme A (propionyl-CoA). Supplementation with sources of propionyl-CoA, including cholesterol, abrogated the restrictive effect of ATRA. This work demonstrates that targeting the coupled metabolism of Mtb and the macrophage improves control of infection and that it is possible to genetically map the mode of bacterial death using CRISPR interference. IMPORTANCE Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is a leading cause of death due to infectious disease. Improving the immune response to tuberculosis holds promise for fighting the disease but is limited by our lack of knowledge as to how the immune system kills M. tuberculosis. Our research identifies a potent way to make relevant immune cells more effective at fighting M. tuberculosis and then uses paired human and bacterial genomic methods to determine the mechanism of that improved bacterial clearance.

Identification of structurally diverse menaquinone-binding antibiotics with in vivo activity against multidrug-resistant pathogens.

The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action. Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, mode of action due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs). Their predicted products (MBA1-6) were rapidly accessed using a synthetic bioinformatic natural product approach, which relies on bioinformatic structure prediction followed by chemical synthesis. Among these six structurally diverse MBAs, four make up two new MBA structural families. The most potent member of each new family (MBA3, MBA6) proved effective at treating methicillin-resistant Staphylococcus aureus infection in a murine peritonitis-sepsis model. The only conserved feature present in all MBAs is the sequence 'GXLXXXW', which we propose represents a minimum MK-binding motif. Notably, we found that a subset of MBAs were active against Mycobacterium tuberculosis both in vitro and in macrophages. Our findings suggest that naturally occurring MBAs are a structurally diverse and untapped class of mechanistically interesting, in vivo active antibiotics.

Ferritin H deficiency deteriorates cellular iron handling and worsens Salmonella typhimurium infection by triggering hyperinflammation.

Iron is an essential nutrient for mammals as well as for pathogens. Inflammation-driven changes in systemic and cellular iron homeostasis are central for host-mediated antimicrobial strategies. Here, we studied the role of the iron storage protein ferritin H (FTH) for the control of infections with the intracellular pathogen Salmonella enterica serovar Typhimurium by macrophages. Mice lacking FTH in the myeloid lineage (LysM-Cre+/+Fthfl/fl mice) displayed impaired iron storage capacities in the tissue leukocyte compartment, increased levels of labile iron in macrophages, and an accelerated macrophage-mediated iron turnover. While under steady-state conditions, LysM-Cre+/+Fth+/+ and LysM-Cre+/+Fthfl/fl animals showed comparable susceptibility to Salmonella infection, i.v. iron supplementation drastically shortened survival of LysM-Cre+/+Fthfl/fl mice. Mechanistically, these animals displayed increased bacterial burden, which contributed to uncontrolled triggering of NF-κB and inflammasome signaling and development of cytokine storm and death. Importantly, pharmacologic inhibition of the inflammasome and IL-1ß pathways reduced cytokine levels and mortality and partly restored infection control in iron-treated ferritin-deficient mice. These findings uncover incompletely characterized roles of ferritin and cellular iron turnover in myeloid cells in controlling bacterial spread and for modulating NF-κB and inflammasome-mediated cytokine activation, which may be of vital importance in iron-overloaded individuals suffering from severe infections and sepsis.

Discovery of novel nitrogenous heterocyclic-containing quinoxaline-1,4-di-N-oxides as potent activator of autophagy in M.tb-infected macrophages.

As a continuation of our research on antimycobacterial agents, a series of novel quinoxaline-1,4-di-N-oxides (QdNOs) containing various nitrogenous heterocyclic moieties at the R6 position were designed and synthesized. Antimycobacterial activities, as well as the cytotoxic effects, of the compounds were assayed. Four compounds (6b, 6f, 6n, and 6o), characterized by 2-carboxylate ethyl or benzyl ester, 6-imidazolyl or 1,2,4-triazolyl, and a 7-fluorine group, exhibited the most potent antimycobacterial activity against M.tb strain H37Rv (MIC ≤ 0.25 µg/mL) with low toxicity in VERO cells (SI = 169.3-412.1). Compound 6o also exhibited excellent antimycobacterial activity in an M.tb-infected macrophage model and was selected for further exploration of the mode of antimycobacterial action of QdNOs. The results showed that compound 6o was capable of disrupting membrane integrity and disturbing energy homeostasis in M.tb. Furthermore, compound 6o noticeably increased cellular ROS levels and, subsequently, induced autophagy in M.tb-infected macrophages, possibly indicating the pathways of QdNOs-mediated inhibition of intracellular M.tb replication. The in vivo pharmacokinetic (PK) profiles indicated that compounds 6o was acceptably safe and possesses favorable PK properties. Altogether, these findings suggest that compound 6o is a promising antimycobacterial candidate for further research.

Something Old, Something New: Ion Channel Blockers as Potential Anti-Tuberculosis Agents.

Tuberculosis (TB) remains a challenging global health concern and claims more than a million lives every year. We lack an effective vaccine and understanding of what constitutes protective immunity against TB to inform rational vaccine design. Moreover, treatment of TB requires prolonged use of multi-drug regimens and is complicated by problems of compliance and drug resistance. While most Mycobacterium tuberculosis (Mtb) bacilli are quickly killed by the drugs, the prolonged course of treatment is required to clear persistent drug-tolerant subpopulations. Mtb's differential sensitivity to drugs is, at least in part, determined by the interaction between the bacilli and different host macrophage populations. Therefore, to design better treatment regimens for TB, we need to understand and modulate the heterogeneity and divergent responses that Mtb bacilli exhibit within macrophages. However, developing drugs de-novo is a long and expensive process. An alternative approach to expedite the development of new TB treatments is to repurpose existing drugs that were developed for other therapeutic purposes if they also possess anti-tuberculosis activity. There is growing interest in the use of immune modulators to supplement current anti-TB drugs by enhancing the host's antimycobacterial responses. Ion channel blocking agents are among the most promising of the host-directed therapeutics. Some ion channel blockers also interfere with the activity of mycobacterial efflux pumps. In this review, we discuss some of the ion channel blockers that have shown promise as potential anti-TB agents.

Bioenergetic Heterogeneity in Mycobacterium tuberculosis Residing in Different Subcellular Niches.

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

Retinoic acid induces antimicrobial peptides and cytokines leading to Mycobacterium tuberculosis elimination in airway epithelial cells.

Tuberculosis (TB) is the leading cause of death by a single infectious agent, Mycobacterium tuberculosis (Mtb). Alveolar macrophages and respiratory epithelial cells are the first cells exposed to Mtb during the primary infection, once these cells are activated, secrete cytokines and antimicrobial peptides that are associated with the Mtb contention and elimination. Vitamins are micronutrients that function as boosters on the innate immune system, however, is unclear whether they have any protective activity during Mtb infection. Thus, we investigated the role of vitamin A (retinoic acid), vitamin C (ascorbic acid), vitamin D (calcitriol), and vitamin E (alfa-tocopherol) as inductors of molecules related to mycobacterial infection in macrophages and epithelial cells. Our results showed that retinoic acid promotes the expression of pro- and anti-inflammatory molecules such as Thymic stromal lymphopoietin (TSLP), ß-defensin-2, IL-1ß, CCL20, ß-defensin-3, Cathelicidin LL-37, TGF-ß, and RNase 7, whereas calcitriol, ascorbic acid, and α-tocopherol lead to an anti-inflammatory response. Treatment of Mtb-infected epithelial cells and macrophage-like cells with the vitamins showed a differential response, where calcitriol reduced Mtb in macrophages, while retinoic acid reduced infection in epithelial cells. Thereby, we propose that a combination of calcitriol and retinoic acid supplementation can drive the immune response, and promotes the Mtb elimination by increasing the expression of antimicrobial peptides and cytokines, while simultaneously modulating inflammation.

Steroid hormone modulates the production of cathelicidin and human ß-defensins in lung epithelial cells and macrophages promoting Mycobacterium tuberculosis killing.

Several studies have documented the interaction between the immune and endocrine systems as an effective defense strategy against tuberculosis, involving the production of several molecules and immunological processes. In this study, we determined the effect of cortisol and dehydroepiandrosterone (DHEA) on the production of antimicrobial peptides such as cathelicidin and human ß-defensin (HBD) -2, and HBD-3 and their effect on intracellular growth of Mycobacterium tuberculosis (Mtb) in lung epithelial cells and macrophages. Our results showed that DHEA promotes the production of these antimicrobial peptides in infected cells, correlating with the decrease of Mtb bacilli loads. These results suggest the use of exogenous DHEA as an adjuvant for tuberculosis therapy.

Curcuma longa L. helps macrophages to control opportunistic micro-organisms during host-microbe interactions.

Aim: Plant products have been evaluated to control opportunistic micro-organisms, as well as fortify immune system cells. Thus, Curcuma longa L. (turmeric) extract was evaluated in interactions of murine macrophages (RAW 264.7) with Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, in order to establish cooperation with defense cells. Materials & methods: Effects of minimal inhibitory concentrations (MIC) of the plant extract were analyzed on phagocytosis, cell viability of RAW 264.7 and production of inflammation-related molecules (IL-1ß, TNF-α, IL-10 and NO). Results: The plant extract was cytocompatible and promoted significant reductions of micro-organisms, and synthesis of inflammation-related molecules, during interactions. Conclusion:C. longa L. extract showed significant antimicrobial response and cooperation with macrophages, by fighting bacteria and yeasts during host-microbe interactions.

Human Mesenchymal Stromal Cell Secretome Promotes the Immunoregulatory Phenotype and Phagocytosis Activity in Human Macrophages.

Human mesenchymal stromal/stem cells (hMSCs) show great promise in cell therapy due to their immunomodulatory properties. The overall immunomodulatory response of hMSCs resembles the resolution of inflammation, in which lipid mediators and regulatory macrophages (Mregs) play key roles. We investigated the effect of hMSC cell-cell contact and secretome on macrophages polarized and activated toward Mreg phenotype. Moreover, we studied the effect of supplemented polyunsaturated fatty acids (PUFAs): docosahexaenoic acid (DHA) and arachidonic acid, the precursors of lipid mediators, on hMSC immunomodulation. Our results show that unlike hMSC cell-cell contact, the hMSC secretome markedly increased the CD206 expression in both Mreg-polarized and Mreg-activated macrophages. Moreover, the secretome enhanced the expression of programmed death-ligand 1 on Mreg-polarized macrophages and Mer receptor tyrosine kinase on Mreg-activated macrophages. Remarkably, these changes were translated into improved Candida albicans phagocytosis activity of macrophages. Taken together, these results demonstrate that the hMSC secretome promotes the immunoregulatory and proresolving phenotype of Mregs. Intriguingly, DHA supplementation to hMSCs resulted in a more potentiated immunomodulation with increased CD163 expression and decreased gene expression of matrix metalloproteinase 2 in Mreg-polarized macrophages. These findings highlight the potential of PUFA supplementations as an easy and safe method to improve the hMSC therapeutic potential.

Xijiao Dihuang decoction improves prognosis of sepsis via inhibition of aerobic glycolysis.

Aerobic glycolysis is a key factor to aggravate progression of sepsis. Xijiao Dihuang decoction (XJDHT) has been proven to have favorable therapeutic effects on sepsis. Our previous study has shown that XJDHT is capable of improving survival from sepsis. In this study we investigated the effects of XJDHT on aerobic glycolysis. The rats were randomly divided into five groups, which included control group, model group, TAK-242 group, XJDHT (25 g/kg) group and XJDHT (12.5 g/kg) group. The contents of cytokines increased in the model group compared with control group, while XJDHT reduced expressions of cytokines. Furthermore, the expressions of TLR4, HIF-1α and PKM2 were reduced significantly in the XJDHT group compared with the model group. There were five groups, including control group, LPS group, siTLR4 group, XJDHT (4 mg/mL) group and XJDHT (2 mg/mL) group in vitro experiments. The IL-1ß and IL-6 were elevated significantly after LPS stimulation in the model group, while XJDHT reduced the expression of cytokines. Protein expressions of TLR4, HIF-1α and PKM2 were increased significantly by stimulation of LPS, while XJDHT down-regulated the expressions of key molecules in the signaling pathway. To conclude, our study implies that XJDHT is capable of improving the prognosis of sepsis by inhibiting aerobic glycolysis via down-regulation of TLR4/HIF-1α/PKM2 signaling pathway.

N6-methylated adenine on the target sites of mamA from Mycobacterium bovis BCG enhances macrophage activation by CpG DNA in mice.

CpG motifs in DNA sequences are recognized by Toll-like receptor 9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine [5 mC]) and modified adenine bases (6-methyladenine [6 mA]). 5 mC inhibits immune activation by CpG DNA; however, it is unclear whether 6 mA inhibits immune activation by CpG DNA. Mycobacterium bovis BCG (BCG) has three adenine methyltransferases (MTases) that act on specific target sequences. In this study, we examined whether the 6 mA at the target sites of adenine MTases affected the immunostimulatory activity of CpG DNA. Our results showed that only 6 mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced interleukin (IL)-12p40 production from murine bone marrow-derived macrophages (BMDMs) stimulated with CpG DNA. Enhancement of IL-12p40 production in BMDMs was also observed when BMDMs were stimulated with CpG DNA ligated to oligodeoxynucleotides (ODNs) harboring 6 mA. Accordingly, we then evaluated whether gDNA from adenine MTase-deficient BCG was less efficient with regard to stimulation of BMDMs. Indeed, gDNA from a mamA-deficient BCG had less ability to activate BMDMs than that from wild-type BCG. We concluded from these results that adenine methylation on ODNs and bacterial gDNA may enhance immune activity induced by CpG DNA.

High-polyphenol extracts from Sorghum bicolor attenuate replication of Legionella pneumophila within RAW 264.7 macrophages.

Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires' Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via increased tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable cereal crop that thrives in arid environments and is well-suited to continued production in warming climates. Sorghum polyphenols have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. Sorghum high-polyphenol extract (HPE) attenuated L. pneumophila intracellular replication in a dose-dependent manner but did not impair either bacterial replication in rich media or macrophage viability. Moreover, HPE treatment enhanced both TNF and IL-6 secretion from L. pneumophila infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila, likely via increased pro-inflammatory cytokine production. This work reveals commonalities between plant polyphenol-mediated antimicrobial activity and provides a foundation for future evaluation of sorghum as an antimicrobial agent.

N-acetylcysteine reduces amphotericin B deoxycholate nephrotoxicity and improves the outcome of murine cryptococcosis.

Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis.

Assaying Chlamydia pneumoniae Persistence in Monocyte-Derived Macrophages Identifies Dibenzocyclooctadiene Lignans as Phenotypic Switchers.

Antibiotic-tolerant persister bacteria involve frequent treatment failures, relapsing infections and the need for extended antibiotic treatment. The virulence of an intracellular human pathogen C. pneumoniae is tightly linked to its propensity for persistence and means for its chemosensitization are urgently needed. In the current work, persistence of C. pneumoniae clinical isolate CV6 was studied in THP-1 macrophages using quantitative PCR and quantitative culture. A dibenzocyclooctadiene lignan schisandrin reverted C. pneumoniae persistence and promoted productive infection. The concomitant administration of schisandrin and azithromycin resulted in significantly improved bacterial eradication compared to sole azithromycin treatment. In addition, the closely related lignan schisandrin C was superior to azithromycin in eradicating the C. pneumoniae infection from the macrophages. The observed chemosensitization of C. pneumoniae was associated with the suppression of cellular glutathione pools by the lignans, implying to a previously unknown aspect of chlamydia-host interactions. These data indicate that schisandrin lignans induce a phenotypic switch in C. pneumoniae, promoting the productive and antibiotic-susceptible phenotype instead of persistence. By this means, these medicinal plant -derived compounds show potential as adjuvant therapies for intracellular bacteria resuscitation.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Drug Discovery Platform Targeting M. tuberculosis with Human Embryonic Stem Cell-Derived Macrophages.

A major limitation in anti-tuberculosis drug screening is the lack of reliable and scalable models for homogeneous human primary macrophage cells of non-cancer origin. Here we report a modified protocol for generating homogeneous populations of macrophage-like cells from human embryonic stem cells. The induced macrophages, referred to as iMACs, presented similar transcriptomic profiles and characteristic immunological features of classical macrophages and were permissive to viral and bacterial infection, in particular Mycobacterium tuberculosis (Mtb). More importantly, iMAC production was amenable to scale up. To evaluate iMAC efficiency in high-throughput anti-tuberculosis drug screening, we performed a phenotypic screening against intracellular Mtb, involving a library of 3,716 compounds that included FDA-approved drugs and other bioactive compounds. Our primary screen identified 120 hits, which were validated in a secondary screen by dose-intracellular and -extracellular Mtb assays. Our confirmatory studies identified a novel anti-Mtb compound, 10-DEBC, also showing activity against drug-resistant strains.