Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide.

Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.

Dual function of LapB (YciM) in regulating Escherichia coli lipopolysaccharide synthesis.

Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new antibiotics. Escherichia coli adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapBcyto). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapBcyto/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapBcyto also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Pseudomonas aeruginosa. Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH.

Common and varied molecular responses of Escherichia coli to five different inhibitors of the lipopolysaccharide biosynthetic enzyme LpxC.

A promising yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides, which are the major constituents of the outer membrane. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the response against five different LpxC inhibitors. All compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that FtsH-mediated proteolysis of the inactivated enzyme is impaired. LpxC inhibition sensitized E. coli to vancomycin and rifampin, which poorly cross the outer membrane of intact cells. Four of the five compounds led to an accumulation of lyso-phosphatidylethanolamine, a cleavage product of phosphatidylethanolamine, generated by the phospholipase PldA. The combined results suggested an imbalance in lipopolysaccharides and phospholipid biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids were consistently induced. Upregulated compound-specific proteins are involved in various functional categories, such as stress reactions, nucleotide, or amino acid metabolism and quorum sensing. Our work shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Moreover, we find that the response of E. coli to LpxC inhibition is distinct from the previously reported response in Pseudomonas aeruginosa.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

Different roles of the heterodimer architecture of galectin-4 in selective recognition of oligosaccharides and lipopolysaccharides having ABH antigens.

The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.

Multiple regulatory inputs including cell envelope stress orchestrate expression of the Escherichia coli rpoN operon.

The rpoN operon, an important regulatory hub in Enterobacteriaceae, includes rpoN encoding sigma factor σ54, hpf involved in ribosome hibernation, rapZ regulating glucosamine-6-phosphate levels, and two genes encoding proteins of the nitrogen-related phosphotransferase system. Little is known about regulatory mechanisms controlling the abundance of these proteins. This study employs transposon mutagenesis and chemical screens to dissect the complex expression of the rpoN operon. We find that envelope stress conditions trigger read-through transcription into the rpoN operon from a promoter located upstream of the preceding lptA-lptB locus. This promoter is controlled by the envelope stress sigma factor E and response regulator PhoP is required for its full response to a subset of stress signals. σE also stimulates ptsN-rapZ-npr expression using an element downstream of rpoN, presumably by interfering with mRNA processing by RNase E. Additionally, we identify a novel promoter in the 3' end of rpoN that directs transcription of the distal genes in response to ethanol. Finally, we show that translation of hpf and ptsN is individually regulated by the RNA chaperone Hfq, perhaps involving small RNAs. Collectively, our work demonstrates that the rpoN operon is subject to complex regulation, integrating signals related to envelope stress and carbon source quality.

Interferon α and ß induce differential transcriptional and functional metabolic phenotypes in human macrophages and blunt glycolysis in response to antigenic stimuli.

The impact of chronic exposure to type I interferons (IFN)-α2a, 2b, and ß on macrophage metabolism, intimately linked to macrophage function, is not well understood. This study assesses the nuanced host responses induced by type I IFN cytokines, offering insights into potential therapeutic approaches in diseases associated with these cytokines. Employing a combination of transcriptional profiling and real-time functional analysis, we delineated metabolic reprogramming in response to chronic IFN exposure. Our results reveal distinct transcriptional metabolic profiles between macrophages chronically exposed to IFN-α and IFN-ß. IFN-ß significantly diminishes the oxygen consumption rate and glycolytic proton extrusion rate in macrophages. Conversely, IFN-α2b decreased parameters of mitochondrial fitness and induced a shift toward glutamine oxidation. Assessing the ability of macrophages to induce glycolysis in response to antigenic stimuli (LPS and iH37Rv), we found that chronic exposure to all IFN subtypes limited glycolytic induction. This study addresses a critical oversight in the literature, where individual roles of IFN subtypes are frequently amalgamated and lack distinction. These findings not only provide novel insights into the divergent effects of IFN-α2a, α2b, and ß on macrophage metabolism but also highlight their potential implications for developing targeted therapeutic strategies.

Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica.

The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing, and agriculture. In this study, we isolated 12 bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica was used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide (LPS), cellulose, and BtuB. Lipopolysaccharide was identified as a susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b which suggested that emergence of LPS mutations in culture with SPLA1a represented collateral sensitivity to SPLA5b. We show that bacteria-phage co-culture with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to single phage co-culture. Identification of host susceptibility and resistance genes and understanding infection dynamics are critical steps in the rationale design of phage cocktails against specific bacterial pathogens.IMPORTANCEAs antibiotic resistance continues to emerge in bacterial pathogens, bacterial viruses (phage) represent a potential alternative or adjunct to antibiotics. One challenge for their implementation is the predisposition of bacteria to rapidly acquire resistance to phages. We describe a functional genomics approach to identify mechanisms of susceptibility and resistance for newly isolated phages that infect and lyse Salmonella enterica and use this information to identify phage combinations that exploit collateral sensitivity, thus increasing efficacy. Collateral sensitivity is a phenomenon where resistance to one class of antibiotics increases sensitivity to a second class of antibiotics. We report a functional genomics approach to rationally design a phage combination with a collateral sensitivity dynamic which resulted in increased efficacy. Considering such evolutionary trade-offs has the potential to manipulate the outcome of phage therapy in favor of resolving infection without selecting for escape mutants and is applicable to other virus-host interactions.

Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells.

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.

Humoral responses are enhanced by facilitating B cell viability by Fcrl5 overexpression in B cells.

B cell initial activity is regulated through a balance of activation and suppression mediated by regulatory molecules expressed in B cells; however, the molecular mechanisms underlying this process remain incompletely understood. In this study, we investigated the function of the Fc receptor-like (Fcrl) family molecule Fcrl5, which is constitutively expressed on naïve B cells, in humoral immune responses. Our study demonstrated that B cell-specific overexpression of Fcrl5 enhanced antibody (Ab) production in both T cell-independent type 1 (TI1) and T cell-dependent (TD) responses. Additionally, it promoted effector B cell formation under competitive conditions in TD responses. Mechanistically, in vitro ligation of Fcrl5 by agonistic Abs reduced cell death and enhanced proliferation in lipopolysaccharide (LPS)-stimulated B cells. In the presence of anti-CD40 Abs and IL-5, the Fcrl5 ligation not only suppressed cell death but also enhanced differentiation into plasma cells. These findings reveal a novel role of Fcrl5 in promoting humoral immune responses by enhancing B cell viability and plasma cell differentiation.

Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury.

Approximately 60% of septic patients developed acute kidney injury (AKI). The mortality rate of septic AKI (SA-AKI) is two to three times higher than that of septic without AKI (SA-non-AKI). The actual functions and mechanisms of CircRNAs in the pathophysiology of SA-AKI remain incompletely understood. Herein, we observed that the mmu_Circ_26986 could be induced by lipopolysaccharide (LPS) and cecum ligation and puncture (CLP) in BUMPT cell line and C57BL/6 mouse kidney, respectively. Functionally, mmu_Circ_26986 suppressed BUMPT cell apoptosis induced by LPS. Mechanistically, mmu_Circ_26986 sponged miRNA-29b-1-5p to upregulate the expression of PAK7. Overexpression of mmu_Circ_26986 ameliorated the progression of CLP-stimulated AKI through miRNA-29b-1-5p/PAK7 axis. In addition, we found that hsa_Circ_0072463, homologous to mmu_Circ_26986, suppressed LPS-induced HK-2 cells apoptosis via regulation of miRNA-29b-1-5p/PAK7 axis. Furthermore, sepsis patients with AKI had a higher level of hsa_Circ_0072463 compared to those without AKI. The sensitivity, specificity and AUC of hsa_Circ_0072463 were 78.8%, 87.9% and 0.866, respectively. Spearman's test indicated a noticeable positive correlation between plasma hsa_Circ_0072463 and serum creatinine in sepsis patients (r = 0.725). In summary, this study reveals that the mmu_Circ_26986/hsa_Circ_0072463 miRNA-29b-1-5p/PAK7 axis mediates septic AKI, and hsa_Circ_0072463 is a potential diagnostic marker for septic AKI.

PmrAB, the two-component system of Acinetobacter baumannii, controls the phosphoethanolamine modification of lipooligosaccharide in response to metal ions.

Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.

Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth.

Salmonella enterica serovar Typhimurium (S. Typhimurium) controls lipopolysaccharide (LPS) biosynthesis by regulating proteolysis of LpxC, the rate-limiting enzyme and target of preclinical antibiotics. PbgA/YejM/LapC regulates LpxC levels and controls outer membrane (OM) LPS composition at the log-to-stationary phase transition. Suppressor substitutions in LPS assembly protein B (LapB/YciM) rescue the LPS and OM integrity defects of pbgA-mutant S. Typhimurium. We hypothesized that PbgA regulates LpxC proteolysis by controlling LapB's ability to bind LpxC as a function of the growth phase. According to existing models, when nutrients are abundant, PbgA binds and restricts LapB from interacting with LpxC and FtsH, which limits LpxC proteolysis. However, when nutrients are limited, there is debate whether LapB dissociates from PbgA to bind LpxC and FtsH to enhance degradation. We sought to examine these models and investigate how the structure of LapB enables salmonellae to control LpxC proteolysis and LPS biosynthesis. Salmonellae increase LapB levels during the stationary phase to promote LpxC degradation, which limits lipid A-core production and increases their survival. The deletion of lapB, resulting in unregulated lipid A-core production and LpxC overabundance, leads to bacterial growth retardation. Tetratricopeptide repeats near the cytosol-inner membrane interface are sufficient for LapB to bind LpxC, and remarkably, LapB and PbgA interact in both growth phases, yet LpxC only associates with LapB in the stationary phase. Our findings support that PbgA-LapB exists as a constitutive complex in S. Typhimurium, which differentially binds LpxC to control LpxC proteolysis and limit lipid A-core biosynthesis in response to changes in the environment.IMPORTANCEAntimicrobial resistance has been a costly setback for human health and agriculture. Continued pursuit of new antibiotics and targets is imperative, and an improved understanding of existing ones is necessary. LpxC is an essential target of preclinical trial antibiotics that can eliminate multidrug-resistant Gram-negative bacterial infections. LapB is a natural LpxC inhibitor that targets LpxC for degradation and limits lipopolysaccharide production in Enterobacteriaceae. Contrary to some studies, findings herein support that LapB remains in complex instead of dissociating from its presumed negative regulator, PbgA/YejM/LapC, under conditions where LpxC proteolysis is enhanced. Advanced comprehension of this critical protein-lipid signaling network will lead to future development and refinement of small molecules that can specifically interfere.

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

Bacterial lipopolysaccharide forms aggregates with apolipoproteins in male and female rat brains after ethanol binges.

Alcohol binge drinking allows the translocation of bacterial lipopolysaccharide (LPS) from the gut to the blood, which activates the peripheral immune system with consequences in neuroinflammation. A possible access/direct signaling of LPS to/in the brain has not yet been described under alcohol abuse conditions. Apolipoproteins are compounds altered by alcohol with high affinity to LPS which may be involved in its transport to the brain or in its elimination. Here, we explored the expression of small components of LPS, in its free form or bound to apolipoproteins, in the brain of female and male rats exposed to alcohol binges. Animals received ethanol oral gavages (3 g/kg every 8 h) for 4 days. LPS or its components (Lipid A and core), LPS-binding protein, corticosterone, lipoproteins (HDL, LDL), apolipoproteins (ApoAI, ApoB, and ApoE), and their receptors were measured in plasma and/or in nonperfused prefrontal cortex (PFC) and cerebellum. Brain LipidA-apolipoprotein aggregates were determined by Western blotting and confirmed by co-immunoprecipitation. In animals exposed to alcohol binges: 1) plasma LPS-binding protein was elevated in both sexes; 2) females showed elevations in plasma ApoAI and corticosterone levels; 3) Lipid A formed aggregates with ApoAI in the female PFC and with ApoB in males, the latter showing Toll-like receptor 4 upregulation in PFC but not females. These results suggest that small bacterial components are present within the brain, forming aggregates with different apolipoproteins, depending on the sex, after alcohol binge intoxications. Results may have implications for the crosstalk between alcohol, LPS, and neuroinflammation.

ApoE4 dysregulation incites depressive symptoms and mitochondrial impairments in mice.

Apolipoprotein E4 (ApoE4) is involved in the stress-response processes and is hypothesized to be a risk factor for depression by means of mitochondrial dysfunction. However, their exact roles and underlying mechanisms are largely unknown. ApoE4 transgenic mice (B6. Cg-ApoEtm1Unc Cdh18Tg( GFAP-APOE i4)1Hol /J) were subjected to stress (lipopolysaccharides, LPS) to elucidate the aetiology of ApoE4-induced depression. LPS treatment significantly aggravated depression-like behaviours, concurrent with neuroinflammation and impaired mitochondrial changes, and melatonin/Urolithin A (UA) + 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) reversed these effects in ApoE4 mice. Concurrently, ApoE4 mice exhibited mitophagy deficits, which could be further exacerbated by LPS stimulation, as demonstrated by reduced Atg5, Beclin-1 and Parkin levels, while PINK1 levels were increased. However, these changes were reversed by melatonin treatment. Additionally, proteomic profiling suggested mitochondria-related signalling and network changes in ApoE4 mice, which may underlie the exaggerated response to LPS. Furthermore, HEK 293T cells transfected with ApoE4 showed mitochondria-associated protein and mitophagy defects, including PGC-1α, TFAM, p-AMPKα, PINK1 and LC3B impairments. Additionally, it aggravates mitochondrial impairment (particularly mitophagy), which can be attenuated by triggering autophagy. Collectively, ApoE4 dysregulation enhanced depressive behaviour upon LPS stimulation.

LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling.

Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification.

Proteomic analysis and protein structure prediction of Shigella phage Sfk20 based on a comparative study using structure prediction approaches.

Bacteriophages are the natural predators of bacteria and are available abundantly everywhere in nature. Lytic phages can specifically infect their bacterial host (through attachment to the receptor) and use their host replication machinery to replicate rapidly, a feature that enables them to kill a disease-causing bacteria. Hence, phage attachment to the host bacteria is the first important step of the infection process. It is reported in this study that the receptor could be an LPS which is responsible for the attachment of the Sfk20 phage to its host (Shigella flexneri 2a). Phage Sfk20 bacteriolytic activity was examined for preliminary optimization of phage titer. The phage Sfk20 viability at different saline conditions was conducted. The LC-MS/MS technique used here for detecting and identifying 40 Sfk20 phage proteins helped us to get an initial understanding of the structural landscape of phage Sfk20. From the identified proteins, six structurally significant proteins were selected for structure prediction using two neural network systems: AlphaFold2 and ESMFold, and one homology modeling software: Phyre2. Later the performance of these modeling systems was compared using various metrics. We conclude from the available and generated information that AlphaFold2 and Phyre2 perform better than ESMFold for predicting Sfk20 phage protein structures.

Low-grade systemic inflammation stimulates microglial turnover and accelerates the onset of Alzheimer's-like pathology.

Several in vivo studies have shown that systemic inflammation, mimicked by LPS, triggers an inflammatory response in the CNS, driven by microglia, characterized by an increase in inflammatory cytokines and associated sickness behavior. However, most studies induce relatively high systemic inflammation, not directly compared with the more common low-grade inflammatory events experienced in humans during the life course. Using mice, we investigated the effects of low-grade systemic inflammation during an otherwise healthy early life, and how this may precondition the onset and severity of Alzheimer's disease (AD)-like pathology. Our results indicate that low-grade systemic inflammation induces sub-threshold brain inflammation and promotes microglial proliferation driven by the CSF1R pathway, contrary to the effects caused by high systemic inflammation. In addition, repeated systemic challenges with low-grade LPS induce disease-associated microglia. Finally, using an inducible model of AD-like pathology (Line 102 mice), we observed that preconditioning with repeated doses of low-grade systemic inflammation, prior to APP induction, promotes a detrimental effect later in life, leading to an increase in Aß accumulation and disease-associated microglia. These results support the notion that episodic low-grade systemic inflammation has the potential to influence the onset and severity of age-related neurological disorders, such as AD.

Tamoxifen exerts direct and microglia-mediated effects preventing neuroinflammatory changes in the adult mouse hippocampal neurogenic niche.

Tamoxifen-inducible systems are widely used in research to control Cre-mediated gene deletion in genetically modified animals. Beyond Cre activation, tamoxifen also exerts off-target effects, whose consequences are still poorly addressed. Here, we investigated the impact of tamoxifen on lipopolysaccharide (LPS)-induced neuroinflammatory responses, focusing on the neurogenic activity in the adult mouse dentate gyrus. We demonstrated that a four-day LPS treatment led to an increase in microglia, astrocytes and radial glial cells with concomitant reduction of newborn neurons. These effects were counteracted by a two-day tamoxifen pre-treatment. Through selective microglia depletion, we elucidated that both LPS and tamoxifen influenced astrogliogenesis via microglia mediated mechanisms, while the effects on neurogenesis persisted even in a microglia-depleted environment. Notably, changes in radial glial cells resulted from a combination of microglia-dependent and -independent mechanisms. Overall, our data reveal that tamoxifen treatment per se does not alter the balance between adult neurogenesis and astrogliogenesis but does modulate cellular responses to inflammatory stimuli exerting a protective role within the adult hippocampal neurogenic niche.