Bacterial Cell Wall Components Research Articles

Lactococcus lactis subsp. cremoris C60 Upregulates Macrophage Function by Modifying Metabolic Preference in Enhanced Anti-Tumor Immunity

Lactococcus lactis subsp. cremoris C60 is a probiotic strain of lactic acid bacteria (LAB) which induces various immune modifications in myeloid lineage cells. These modifications subsequently regulate T cell function, resulting in enhanced immunity both locally and systemically. Here, we report that C60 suppresses tumor growth by enhancing macrophage function via metabolic alterations, thereby increasing adenosine triphosphate (ATP) production in a murine melanoma model. Intragastric (i.g.) administration of C60 significantly reduced tumor volume compared to saline administration in mice. The anti-tumor function of intratumor (IT) macrophage was upregulated in mice administered with C60, as evidenced by an increased inflammatory phenotype (M1) rather than an anti-inflammatory/reparative (M2) phenotype, along with enhanced antigen-presenting ability, resulting in increased tumor antigen-specific CD8+ T cells. Through this functional modification, we identified that C60 establishes a glycolysis-dominant metabolism, rather than fatty acid oxidation (FAO), in IT macrophages, leading to increased intracellular ATP levels. To address the question of why orally supplemented C60 exhibits functions in distal places, we found a possibility that bacterial cell wall components, which could be distributed throughout the body from the gut, may induce stimulatory signals in peripheral macrophages via Toll-like receptors (TLRs) signaling activation. Thus, C60 strengthens macrophage anti-tumor immunity by promoting a predominant metabolic shift towards glycolysis upon TLR-mediated stimulation, thereby increasing substantial energy production.

Peptidoglycan modulation by Francisella tularensis

HAYDEN HESS, STUART CANTLAY, JOSEPH HORZEMPA, Department of Biomedical Sciences, West Liberty University, West Liberty, WV 26074 and MELANIE SAL, Department of Biology, West Virginia Wesleyan College, Buckhannon, WV 26201. Peptidoglycan modulation by Francisella tularensis. Peptidoglycan, a crucial component of bacterial cell walls, plays a fundamental role in maintaining cell shape, integrity, and resistance to osmotic pressure. Mycoplasmas are the only bacteria that do not produce peptidoglycan cell walls. Other peptidoglycan-producing bacteria are capable of transiently existing without a peptidoglycan as an “L-form”, but only on solid media. Preliminary observations made by our laboratory suggested that Francisella tularensis bacteria do not produce detectible levels of peptidoglycan while growing in liquid media. However, increasing salt concentration induces the production of peptidoglycan in all bacteria. In this study we confirmed and extended these observations by incubating bacteria with fluorescent D-alanine (which integrates into the peptidoglycan during synthesis). Fluorescence microscopy revealed that very few F. tularensis bacteria produce detectable peptidoglycan in low salt media, while seemingly all individual bacteria produce this layer in high salt media. Interestingly, the genome of F. tularensis encodes two distinct beta-lactamases. Here we show that both the wild-type F. tularensis LVS and beta-lactamase null mutant were both resistant to high levels of beta-lactam antibiotics in low salt liquid media. However, only the wild type and complemented mutant strains were resistant to beta-lactam antibiotics in high salt media while the beta lactamase null mutant was susceptible. These data further support the conclusion that F. tularensis does not produce, nor need, peptidoglycan in low salt liquid media. Future studies will investigate whether peptidoglycan modulation is required to avoid detection by the innate immune system.

MiRNA-Mediated Fine Regulation of TLR-Induced M1 Polarization.

Macrophage polarization to the M1 spectrum is induced by bacterial cell wall components through stimulation of Toll-like family (TLR) receptors. By orchestrating the expression of relevant mediators of the TLR cascade, as well as associated pathways and feedback loops, macrophage polarization is coordinated to ensure an appropriate immune response. This is central to the successful control of pathogens and the maintenance of health. Macrophage polarization is known to be modulated at both the transcriptional and post-transcriptional levels. In recent years, the miRNA-based post-transcriptional regulation of M1 polarization has received increasing attention from the scientific community. Comparative studies have shown that TLR stimulation alters the miRNA profile of macrophages and that macrophages from the M1 or the M2 spectrum differ in terms of miRNAs expressed. Simultaneously, miRNAs are considered critical post-transcriptional regulators of macrophage polarization. In particular, miRNAs are thought to play a regulatory role in the switch between the early proinflammatory response and the resolution phase. In this review, we will discuss the current state of knowledge on the complex interaction of transcriptional and post-transcriptional regulatory mechanisms that ultimately determine the functionality of macrophages.

Multi-Omics Study on Molecular Mechanisms of Single-Atom Fe-Doped Two-Dimensional Conjugated Phthalocyanine Framework for Photocatalytic Antibacterial Performance.

Currently, photocatalysis of the two-dimensional (2D) conjugated phthalocyanine framework with a single Fe atom (CPF-Fe) has shown efficient photocatalytic activities for the removal of harmful effluents and antibacterial activity. Their photocatalytic mechanisms are dependent on the redox reaction-which is led by the active species generated from the photocatalytic process. Nevertheless, the molecular mechanism of CPF-Fe antimicrobial activity has not been sufficiently explored. In this study, we successfully synthesized CPF-Fe with great broad-spectrum antibacterial properties under visible light and used it as an antibacterial agent. The molecular mechanism of CPF-Fe against Escherichia coli and Salmonella enteritidis was explored through multi-omics analyses (transcriptomics and metabolomics correlation analyses). The results showed that CPF-Fe not only led to the oxidative stress of bacteria by generating large amounts of h+ and ROS but also caused failure in the synthesis of bacterial cell wall components as well as an osmotic pressure imbalance by disrupting glycolysis, oxidative phosphorylation, and TCA cycle pathways. More surprisingly, CPF-Fe could disrupt the metabolism of amino acids and nucleic acids, as well as inhibit their energy metabolism, resulting in the death of bacterial cells. The research further revealed the antibacterial mechanism of CPF-Fe from a molecular perspective, providing a theoretical basis for the application of CPF-Fe photocatalytic antibacterial nanomaterials.

Peptidoglycan in osteoarthritis synovial tissue is associated with joint inflammation

ObjectivesPeptidoglycan (PG) is an arthritogenic bacterial cell wall component whose role in human osteoarthritis is poorly understood. The purpose of this study was to determine if PG is present in synovial tissue of osteoarthritis patients at the time of primary total knee arthroplasty (TKA), and if its presence is associated with inflammation and patient reported outcomes.MethodsIntraoperative synovial tissue and synovial fluid samples were obtained from 56 patients undergoing primary TKA, none of whom had history of infection. PG in synovial tissue was detected by immunohistochemistry (IHC) and immunofluorescence microscopy (IFM). Synovial tissue inflammation and fibrosis were assessed by histopathology and synovial fluid cytokine quantification. Primary human fibroblasts isolated from arthritis synovial tissue were stimulated with PG to determine inflammatory cytokine response.ResultsA total of 33/56 (59%) of primary TKA synovial tissue samples were positive for PG by IHC, and PG staining colocalized with markers of synovial macrophages and fibroblasts by IFM. Synovial tissue inflammation and elevated IL-6 in synovial fluid positively correlated with PG positivity. Primary human fibroblasts stimulated with PG secreted high levels of IL-6, consistent with ex vivo findings. Interestingly, we observed a significant inverse correlation between PG and age at time of TKA, indicating younger age at time of TKA was associated with higher PG levels.ConclusionPeptidoglycan is commonly found in synovial tissue from patients undergoing TKA. Our data indicate that PG may play an important role in inflammatory synovitis, particularly in patients who undergo TKA at a relatively younger age.

Lipoteichoic acids influence cell shape and bacterial division of Streptococcus suis serotype 2, but play a limited role in the pathogenesis of the infection

Streptococcus suis serotype 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans, responsible for substantial economic losses to the swine industry worldwide. The pathogenesis of infection and the role of bacterial cell wall components in virulence have not been fully elucidated. Lipoproteins, peptidoglycan, as well as lipoteichoic acids (LTA) have all been proposed to contribute to virulence. In the present study, the role of the LTA in the pathogenesis of the infection was evaluated through the characterisation of a mutant of the S. suis serotype 2 strain P1/7 lacking the LtaS enzyme, which mediates the polymerization of the LTA poly-glycerolphosphate chain. The ltaS mutant was confirmed to completely lack LTA and displayed significant morphological defects. Although the bacterial growth of this mutant was not affected, further results showed that LTA is involved in maintaining S. suis bacterial fitness. However, its role in the pathogenesis of the infection appears limited. Indeed, LTA presence reduces self-agglutination, biofilm formation and even dendritic cell activation, which are important aspects of the pathogenesis of the infection caused by S. suis. In addition, it does not seem to play a critical role in virulence using a systemic mouse model of infection.

The altered TBI fecal microbiome is stable and functionally distinct.

Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

A CTL − Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections

BackgroundGut bacteria are beneficial to the host, many of which must be passed on to host offspring. During metamorphosis, the midgut of holometabolous insects undergoes histolysis and remodeling, and thus risks losing gut bacteria. Strategies employed by holometabolous insects to minimize this risk are obscure. How gut bacteria affect host insects after entering the hemocoel and causing opportunistic infections remains largely elusive.ResultsWe used holometabolous Helicoverpa armigera as a model and found low Lactobacillus load, high level of a C-type lectin (CTL) gene CD209 antigen-like protein 2 (CD209) and its downstream lysozyme 1 (Lys1) in the midgut of the wandering stage. CD209 or Lys1 depletion increased the load of midgut Lactobacillus, which further translocate to the hemocoel. In particular, CD209 or Lys1 depletion, injection of Lactobacillus plantarum, or translocation of midgut L. plantarum into the hemocoel suppressed 20-hydroxyecdysone (20E) signaling and delayed pupariation. Injection of L. plantarum decreased triacylglycerol and cholesterol storage, which may result in insufficient energy and 20E available for pupariation. Further, Lysine-type peptidoglycan, the major component of gram-positive bacterial cell wall, contributed to delayed pupariation and decreased levels of triacylglycerols, cholesterols, and 20E, in both H. armigera and Drosophila melanogaster.ConclusionsA mechanism by which (Lactobacillus-induced) opportunistic infections delay insect metamorphosis was found, namely by disturbing the homeostasis of lipid metabolism and reducing 20E production. Moreover, the immune function of CTL − Lys was characterized for insect metamorphosis by maintaining gut homeostasis and limiting the opportunistic infections.

Visible light-enhancing antibacterial ability of gold ions for its application of the prevention and treatment of dentin caries

To develop new antimicrobial strategy or agents has always been an eternal topic for the researchers. In this study, we have found that the visible light can enhance the antibacterial action of gold ions because of the photo-reduction and thus the visible light energy can be used to kill bacteria. The electron accepting ability of Au ions exposed to the visible light can strengthen, so their damage capability for the organic compounds from bacteria simultaneously enhances. The products of light-triggering Au ions, Au nanoparticles (Au NPs), also can bind protein and other bacterial cell wall components to further enhance the antibacterial ability. With the aid of the penetrating capacity of Au ions and light, light can also enhance the antibacterial action of Au ion in the biofilm and dentinal tubules and their products have the ability to crosslink the collagens to protect them from the degradation of collagenase. The toxicity of Au ions limits its application in clinic treatment, but the toxicity can decrease significantly after Au ions kills bacteria and the light can further reduce the toxicity as well. High concentration of Au ions and long-exposure light for dentin-pulp complex also are safe in vivo. Hence, light-enhancing the antibacterial treatment of Au ions has the potential to be used in the prevention and cure of the caries.

In silico strategies for identifying therapeutic candidates against Acinetobacter baumannii: spotlight on the UDP-N-acetylmuramoyl-L-alanine-D-glutamate:meso-diaminopimelate ligase (MurE)

The opportunistic bacterium Acinetobacter baumannii, which belongs to ESKAPE group of pathogenic bacteria, is leading cause of infections associated with gram-negative bacteria. Acinetobacter baumannii causes severe diseases, such as VAP (ventilator-associated pneumonia), meningitis, and UTI (urinary tract infections) among the nosocomial infections contracted in hospitals. The high infection rate and growing resistance to the vast array of antibiotics makes it paramount to look for new therapeutic strategies against this pathogen. The most promising therapeutic targets are the proteins involved in the synthesis of peptidoglycan which is chief component of bacterial cell wall, MurE is one of those enzymes and is responsible for the addition of one unit of meso-diaminopimelic acid (meso-A2pm) to the nucleotide precursor, UDPMurNAc-L-Ala-D-Glu, and aids in the formation of crosslinker pentapeptide chain. The three-dimensional structure of MurE was modelled using homology modelling technique and then vHTS was performed using this model against Approved Drug Library on DrugRep server using AutoDock Vina. Out of 500 drug molecules, two were selected based on estimated binding affinity, interaction pattern, interacting residues, etc. The selected drug molecules are DB12887 (Tazemetostat) and DB13879 (Glecaprevir). Then, MD simulations were performed on native MurE and its complexes with ligands to examine their dynamical behaviour, stability, integrity, compactness, and folding properties. The protein-ligand complexes were then subjected to binding free energy calculations using the MM/PBSA-based binding free energy analysis and the values are −109.788 ± 8.03 and −152.753 ± 11.98 kcal for MurE-DB12887 and MurE-DB13879 complex, respectively. All the analysis performed on MD trajectories for the complexes of these ligands with protein provided plenty of dependable evidences to consider these molecules for inhibition of MurE enzyme from A. baumannii. Communicated by Ramaswamy H. Sarma

Inhaled endotoxin induces a systemic neutrophil response without affecting cardiovascular measures in a randomized cross-over exposure study

Objective The gram-negative bacterial cell wall component endotoxin (lipopolysaccharide, LPS) is a key component of particulate matter (PM). PM exposure is associated with cardiovascular morbidity and mortality. However, the contribution of individual components of PM to acute and chronic cardiovascular measures is not clear. This study examines whether systemic inflammation induced by LPS inhalation causes acute changes in cardiovascular physiology measures. Materials and Methods In this double blinded, placebo-controlled crossover study, fifteen adult volunteers underwent inhalation exposure to 20,000 EU Clinical Center Reference Endotoxin (CCRE). Peripheral blood and induced sputum neutrophils were obtained at baseline and six hours post-exposure. Blood pressure, measures of left ventricular function (ejection fraction (LVEF) and global longitudinal strain (LVGLS)), and indices of endothelial function (flow mediated dilation (FMD) and velocity time integral during hyperemia (VTIhyp)) were measured before and after treatment. Wilcoxon sign-rank tests and linear mixed models were used for statistical analysis. Results In comparison with normal saline, LPS inhalation resulted in significant increases in peripheral blood and sputum neutrophils but was not associated with significant alterations in blood pressure, LVGLS, LVEF, FMD, or VTIhyp. Discussion and Conclusions In healthy adults, systemic inflammation after LPS inhalation was not associated with acute changes in cardiovascular physiology. Larger studies are needed to investigate the effects of other PM components on inflammation induced cardiovascular dysfunction.

Insect Bacteriocytes: Adaptation, Development, and Evolution.

Bacteriocytes are host cells specialized to harbor symbionts in certain insect taxa. The adaptation, development, and evolution of bacteriocytes underlie insect symbiosis maintenance. Bacteriocytes carry enriched host genes of insect and bacterial origin whose transcription can be regulated by microRNAs, which are involved in host-symbiont metabolic interactions. Recognition proteins of peptidoglycan, the bacterial cell wall component, and autophagy regulate symbiont abundance in bacteriocytes. Horizontally transferred genes expressed in bacteriocytes influence the metabolism of symbiont peptidoglycan, which may affect the bacteriocyte immune response against symbionts. Bacteriocytes release or transport symbionts into ovaries for symbiont vertical transmission. Bacteriocyte development and death, regulated by transcriptional factors, are variable in different insect species. The evolutionary origin of insect bacteriocytes remains unclear. Future research should elucidate bacteriocyte cell biology, the molecular interplay between bacteriocyte metabolic and immune functions, the genetic basis of bacteriocyte origin, and the coordination between bacteriocyte function and host biology in diverse symbioses.

Metal-Phenolic Nanocapsules with Photothermal Antibacterial and Ros Scavenging Ability for Diabetic Wound Healing.

The presence of bacteria in diabetic wounds not only leads to the formation of biofilms but also triggers oxidative stress and inflammatory responses, which hinder the wound-healing process. Therefore, it is imperative to formulate a comprehensive strategy that can proficiently eliminate bacteria and enhance the wound microenvironment. Herein, this work develops multifunctional metal-phenolic nanozymes (TA-Fe/Cu nanocapsules), wherein the one-pot coordination of tannic acid (TA)and Fe3+/Cu2+ using a self-sacrificial template afforded hollow nanoparticles (NPs) with exceptional photothermal and reactive oxygen species scavenging capabilities. After photothermal disruption of the biofilms, TA-Fe/Cu NPs autonomously capture bacteria through hydrogen bonding interactions with peptidoglycans (the bacterial cell wall component), ultimately bolstering the bactericidal efficacy. Furthermore, these NPs exhibit peroxidase-like enzymatic activity, efficiently eliminating surplus hydrogen peroxide in the vicinity of the wound and mitigating inflammatory responses. As the wound transitions into the remodeling phase, the presence of Cu2+ stimulates vascular migration and regeneration, expediting the wound-healing process. This study innovatively devises a minimalist approach to synthesize multifunctional metal-phenolic nanozymes integrating potent photothermal antibacterial activity, bacterial capture, anti-inflammatory, and angiogenesis properties, showcasing their great potential for diabetic wound treatment.

Chirality-influenced antibacterial behavior of gold nanoclusters

Chirality exists widely in living systems and plays a crucial role in pharmaceutical production and theranostics. Gold nanoclusters (AuNCs) have recently been developed as potential antibacterial therapeutics, but the influence of chirality on their antibacterial behavior is unexplored. Here we prepare ultrasmall L-, D-, and DL-histidine templated AuNCs (denoted as L-AuHis, D-AuHis, and DL-AuHis, respectively) using a simple method. The three AuNCs possess the same physicochemical properties and maintain the chirality consistent with histidine. Antibacterial assay results using Staphylococcus aureus as a representative demonstrate the chirality-influenced antibacterial behavior of these AuNCs. An in-depth investigation revealed that the antibacterial activity of these chiral AuNCs was mainly attributed to cell structure damage and thiol-redox homeostasis imbalance. D-AuHis exhibit more potent antibacterial activity compared to their enantiomers. This difference in antibacterial behavior is mainly since the L-AuHis and DL-AuHis can react with bacterial cell wall components, such as lipoteichoic acid, to form large-sized nanoparticles, thereby weakening the antibacterial activity compared with the D-AuHis. The antibacterial effect of AuHis was further validated in a bacterial-infected wound model. Our findings provide a deeper insight into the establishment of unique chiral inorganic antibacterial constructions and hint at the potentiality of dextrorotatory AuNCs having strengthened antibiotic activity.

Noncoding GATA2 Genetic Variation As a Determinant of Hematopoietic Stem/Progenitor Cell Activity and Mobilization Efficiency

Germlinecoding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). We generated a mouse model of GATA2 deficiency syndrome by engineering mice with a single-nucleotide variant in an intronic enhancer (+9.5) from GATA2 deficiency patients combined with deletion of the enhancer from the second allele (compound heterozygous; CH). This system models patients harboring a germline pathogenic allele accompanied by epigenetic silencing of the normal allele. Unlike homozygous Gata2 coding or regulatory deletions that are embryonic lethal, CH represents a unique system to analyze postnatal GATA2 function. We demonstrated that adult germline Gata2 variation alters steady-state HSPCs, blocks HSPC expansion and differentiation upon chemotherapy, attenuates long-term repopulating activity following bone marrow transplantation, leads to bone marrow failure following chronic inflammation by the viral mimetic polyI:C, alters HSPC response to the bacterial cell wall component LPS and attenuates HSPC mobilization in response to granulocyte colony stimulating factor (G-CSF) (Soukup et al., 2019, Soukup et al., 2021). As short-term G-CSF treatment induces HSPC metabolic gene signatures (Fast et al., 2021) and expansion and cell cycle entry (Schuettpelz et al., 2014), and CH have decreased MPPs (LSKCD150 -CD48 -) and common myeloid progenitors (CMPs) (Lin −Sca1 −Kit +FcγR −CD34 +) (Soukup et al., 2021), we asked if mobilization defects reflected decreased functional HSPCs in bone marrow. CH had 2.2-fold fewer colony forming units (CFU) from bone marrow (p = 0.007) than wild type (WT), reflecting a paucity of functional HSPCs. 16 hours following a single G-CSF dose, WT bone marrow immunophenotypic HSCs (LSKCD150 +CD48 -) and MPPs expanded 1.8-fold (p = 0.001 and 0.01, respectively); CH HSCs and MPPs were unchanged. CH led to decreased HSPCs and failed to induce rapid HSPC expansion post-G-CSF. We tested whether Gata2 variation impairs mobilization in response to different stimuli, or if select pathways are impacted via disrupting the CXCR4/CXCL12 chemokine axis with the CXCR4 antagonist plerixafor, stromal/neutrophil CXCR2 activation with rhIL-8, or VLA-4/VCAM1 binding with VLA-4 inhibitor BIO5192. IL-8 or BIO5192 resulted in 3.9- and 2.2-fold increased WT CFU, respectively (p &amp;lt; 0.0001 and 0.0014) without altering CH CFU. By contrast, mobilization with plerixafor resulted in a 4.3-fold increase in WT CFU (p &amp;lt; 0.0001) and 4.5-fold increase in CH CFU (p = 0.004). Targeting these pathways collectively with G-CSF was insufficient to restore normal mobilization in CH. Competitive transplantations with peripheral blood revealed G-CSF + plerixafor increased WT donor-derived hematopoiesis 24- and 20-fold at 8 and 12 weeks, respectively, versus vehicle control. Treatment of CH did not increase donor-derived contributions. Since GATA2 deficiency syndrome and somatic GATA2 variation involve heterozygous alleles, we tested whether monoallelic +9.5 Ets motif variation disrupts mobilization. G-CSF increased CFU 28.5-fold (p &amp;lt; 0.0001) relative to vehicle-treated WT. Mobilization of Gata2 Ets +/- HSPCs were reduced significantly (p = 0.003). CH variation severely disrupted mobilization, with 7.3-fold fewer CFU than Gata2 Ets +/- (p = 0.02). Synergistic G-CSF/plerixafor treatment increased CFU 89-fold in WT versus untreated (p &amp;lt; 0.0001), and Gata2 Ets +/- mobilization was reduced (p = 0.006) and not improved relative to Gata2 Ets -/- or CH, resembling G-CSF/BIO5192. Thus, Gata2 heterozygosity impaired G-CSF-induced HSPC mobilization with all regimens. Although GATA2 noncoding variation is not commonly evaluated, analysis of human data will be performed to assess relationships between variation and poor mobilization outcomes. Gata2 variation depletes functional HSPCs in the bone marrow, reducing the available HSPCs to mobilize and rendering most mobilization regimens inefficient. Ongoing studies are elucidating molecular mechanisms underlying the mobilization blockade. Our mobilization-defective system offers unique utility for elucidating fundamental HSPC mechanisms e.g., in the context of additional environmental and/or genetic insults. Future knowledge linking functionality of mobilization regimens in these contexts may unveil insights into mechanisms operational in poor mobilizers.

Identification of potential molecular targets and repurposed drugs for tuberculosis using network-based screening approach, molecular docking, and simulation

The spread of drug-resistant strains of tuberculosis has hampered efforts to control the disease worldwide. The Mycobacterium tuberculosis cell wall envelope is dynamic, with complex features that protect it from the host immunological response. As a result, the bacterial cell wall components represent a potential target for drug discovery. Protein-protein interaction networks (PPIN) are critical for understanding disease conditions and identifying precise therapeutic targets. We used a rational theoretical approach by constructing a PPIN with the proteins involved in cell wall biosynthesis. The PPIN was constructed through the STRING database and embB was identified as a key protein by using four topological measures, betweenness, closeness, degree, and eigenvector, in the CytoNCA tool in Cytoscape. The ‘Drug repurposing’ approach was employed to find suitable inhibitors against embB. We used the Schrödinger suites for molecular docking, molecular dynamics simulation, and binding free energy calculations to validate the binding of protein with the ligand. FDA-approved drugs from the ZINC database and DrugBank were screened against embB (PDB ID: 7BVF) using high-throughput virtual screening, standard precision, and extra precision docking. The drugs were screened based on the XP docking score of the standard drug ethambutol. Accordingly, from the top five hits, azilsartan and dihydroergotamine were selected based on the binding free energy values and were further subjected to Molecular Dynamics Simulation studies for 100 ns. Our study confirms that Azilsartan and Dihydroergotamine form stable complexes with embB and can be used as potential lead molecules based on further in vitro and in vivo experimental validation. Communicated by Ramaswamy H. Sarma

Multifaceted roles and regulation of nucleotide-binding oligomerization domain containing proteins.

Nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2, are cytosolic receptors that recognize dipeptides and tripeptides derived from the bacterial cell wall component peptidoglycan (PGN). During the past two decades, studies have revealed several roles for NODs beyond detecting PGN fragments, including activation of an innate immune anti-viral response, NOD-mediated autophagy, and ER stress induced inflammation. Recent studies have also clarified the dynamic regulation of NODs at cellular membranes to generate specific and balanced immune responses. This review will describe how NOD1 and NOD2 detect microbes and cellular stress and detail the molecular mechanisms that regulate activation and signaling while highlighting new evidence and the impact on inflammatory disease pathogenesis.

Copper infused fabric attenuates inflammation in macrophages.

While inflammation is an important immune response for protection from infections, excessive or prolonged inflammation can lead to a variety of debilitating diseases including skin disease, diabetes, heart disease, stroke, autoimmune diseases and cancer. Inflammation is a graded response that is typically initiated when resident macrophages sense the presence of pathogens or damage in the tissue and produce inflammatory cytokines and chemokines to kill the pathogen, clear debris and dead tissue, and initiate tissue repair. Here we show that copper-infused fabrics can prevent inflammation by blocking the production of inflammatory cytokines from macrophages after being exposed to LPS, a component of bacterial cell wall. Mechanistically, we show that copper-infused fabrics can significantly reduce the NF-κB and IRF3 activation in LPS-stimulated macrophages. Given the importance of excessive inflammation in diabetes, we show that copper can reduce insulin resistance mediated by inflammatory cytokines in muscle cells. Our data show that copper infused fabrics may be useful to reduce excessive inflammation in macrophages and improve insulin sensitivity in skeletal muscles.

Modulation of the immune response by the host defense peptide IDR-1002 in chicken hepatic cell culture

IDR-1002, a synthetic host defense peptide (HDP), appears to be a potential candidate for the treatment of bacterial infections and the consequent inflammatory response due to its potent immunomodulatory activity. This is of relevance to the emerging issue of antimicrobial resistance in the farming sector. In this study, the effects of IDR-1002 were investigated on a chicken hepatocyte‒non-parenchymal cell co-culture, and the results revealed that IDR-1002 had complex effects on the regulation of the hepatic innate immunity. IDR-1002 increased the levels of both RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and Macrophage colony stimulating factor (M-CSF), suggesting the peptide plays a role in the modulation of macrophage differentiation, also reflected by the reduced concentrations of interleukin (IL)-6 and IL-10. The pro-inflammatory cytokine release triggered by the bacterial cell wall component lipoteichoic acid (LTA) was ameliorated by the concomitantly applied IDR-1002 based on the levels of IL-6, chicken chemotactic and angiogenic factor (CXCLi2) and interferon (IFN)-γ. Moreover, the production of nuclear factor erythroid 2-related factor 2 (Nrf2), an essential transcription factor in the antioxidant defense pathway, was increased after IDR-1002 exposure, while protein carbonyl (PC) levels were also elevated. These findings suggest that IDR-1002 affects the interplay of the cellular immune response and redox homeostasis, thus the peptide represents a promising tool in the treatment of bacterially induced inflammation in chickens.

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner

BackgroundLung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host–pathogen interactions during pneumococcal infections.MethodsFollowing in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid.ResultsIn vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment.ConclusionStreptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms.EnamKsCfhZcCbpPUq5E2ztVideo