Dementia exacerbates periodontal bone loss in females.

BACKGROUND: Periodontitis is a chronic inflammatory disease defined by the pathologic loss of the periodontal ligament and alveolar bone in relation to aging. Although clinical cohort studies reported that periodontitis is significantly elevated in males compared to females, emerging evidence indicates that females with dementia are at a greater risk for periodontitis and decreased alveolar bone. OBJECTIVE: This study aimed to evaluate whether dementia is a potential sex-dependent risk factor for periodontal bone loss using an experimental model of periodontitis induced in the triple transgenic (3x-Tg) dementia-like mice and clinical samples collected from senior 65 plus age patients with diagnosed dementia. MATERIALS AND METHODS: We induced periodontitis in dementia-like triple-transgenic (3x-Tg) male and female mice and age-matched wild-type (WT) control mice by ligature placement. Then, alveolar bone loss and osteoclast activity were evaluated using micro-CT and in situ imaging assays. In addition, we performed dental examinations on patients with diagnosed dementia. Finally, dementia-associated Aß42 and p-Tau (T181) and osteoclastogenic receptor activator of nuclear factor kappa-Β ligand (RANKL) in gingival crevicular fluid (GCF) collected from mice and clinical samples were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Alveolar bone loss and in situ osteoclast activity were significantly elevated in periodontal lesions of 3x-Tg females but not males, compared to wild-type control mice. In addition, we also observed that the probing pocket depth (PPD) was also significantly elevated in female patients with dementia. Using ELISA assay, we observed that females had elevated levels of osteoclastogenic RANKL and dementia-associated Aß42 and p-Tau (T181) in the GCF collected from experimental periodontitis lesions and clinical samples. CONCLUSION: Altogether, we demonstrate that females with dementia have an increased risk for periodontal bone loss compared to males.

Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells.

The maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of ASAH1 in OSCC. Because a uniquely structured P. gingivalis-derived phosphoethanolamine dihydroceramide (PEDHC) inhibits the proliferation of normal human fibroblasts, this study aimed to test the effect of PEDHC on the survival of human oral squamous OECM-1 cells in vitro. We demonstrated that the P. gingivalis dihydroceramide-null (ΔPG1780) strain upregulates the expression of ASAH1 mRNA and promotes aggressive proliferation and migration of OECM-1 cells compared to the parent P. gingivalis-W83 strain. In addition, the intracellular concentration of ceramides was dramatically elevated in OECM-1 cells exposed to PEDHC in vitro. Furthermore, PEDHC inhibited expression patterns of ASAH1 mRNA as well as some genes associated with degradation of the basement membranes and extracellular matrix, for example, MMP-2, ADAM-17 and IL-6, in OECM-1 cells. Altogether, these data indicated that PEDHC produced by P. gingivalis inhibits acid ceramidase expression, promotes intracellular ceramide accumulation and suppresses the survival and migration of OSCC cells in vitro. Further studies are needed to determine molecular mechanisms of PEDHC-mediated inhibitory effect(s) on OSCC using in vivo models of oral cancer.

Expression of the 2Duf protein in wild-type Bacillus subtilis spores stabilizes inner membrane proteins and increases spore resistance to wet heat and hydrogen peroxide.

AIMS: This work aimed to characterize spore inner membrane (IM) properties and the mechanism of spore killing by wet heat and H2O2 with spores overexpressing the 2Duf protein, which is naturally encoded from a transposon found only in some Bacillus strains with much higher spore resistance than wild-type spores. METHODS AND RESULTS: Killing of Bacillus subtilis spores by wet heat or hydrogen peroxide (H2O2) was slower when 2Duf was present, and Ca-dipicolinic acid release was slower than killing. Viabilities on rich plates of wet heat- or H2O2 -treated spores +/- 2Duf were lower when NaCl was added, but higher with glucose. Addition of glucose but not Casamino acids addition increased treated spores' viability on minimal medium plates. Spores with 2Duf required higher heat activation for germination, and their germination was more wet-heat resistant than that of wild-type spores, processes that involve IM proteins. IM permeability and lipid mobility were lower in spores with 2Duf, although IM phospholipid composition was similar in spores +/- 2Duf. CONCLUSIONS: These results and previous work suggests that wet heat and H2O2 kill spores by damaging an IM enzyme or enzymes involved in oxidative phosphorylation.

Dihydroceramides Derived from Bacteroidetes Species Sensitize TRPV1 Channels.

Bacterial colonization of open wounds is common, and patients with infected wounds often report significantly elevated pain sensitivity at the wound site. Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels are known to play an important role in pain signaling and may be sensitized under pro-inflammatory conditions. Bacterial membrane components, such as phosphoethanolamine dihydroceramide (PEDHC), phosphoglycerol dihydroceramide (PGDHC), and lipopolysaccharide (LPS), are released in the environment from the Gram-negative bacteria of the Bacteroidetes species colonizing the infected wounds. Here, we used intracellular calcium imaging and patch-clamp electrophysiology approaches to determine whether bacterially derived PEDHC, PGDHC, or LPS can modulate the activity of the TRPV1 channels heterologously expressed in HEK cells. We found that PEDHC and PGDHC can sensitize TRPV1 in a concentration-dependent manner, whereas LPS treatment does not significantly affect TRPV1 activity in HEK cells. We propose that sensitization of TRPV1 channels by Bacteroidetes-derived dihydroceramides may at least in part underlie the increased pain sensitivity associated with wound infections.

In Situ Raman Study of Neurodegenerated Human Neuroblastoma Cells Exposed to Outer-Membrane Vesicles Isolated from Porphyromonas gingivalis.

The aim of this study was to elucidate the chemistry of cellular degeneration in human neuroblastoma cells upon exposure to outer-membrane vesicles (OMVs) produced by Porphyromonas gingivalis (Pg) oral bacteria by monitoring their metabolomic evolution using in situ Raman spectroscopy. Pg-OMVs are a key factor in Alzheimer's disease (AD) pathogenesis, as they act as efficient vectors for the delivery of toxins promoting neuronal damage. However, the chemical mechanisms underlying the direct impact of Pg-OMVs on cell metabolites at the molecular scale still remain conspicuously unclear. A widely used in vitro model employing neuroblastoma SH-SY5Y cells (a sub-line of the SK-N-SH cell line) was spectroscopically analyzed in situ before and 6 h after Pg-OMV contamination. Concurrently, Raman characterizations were also performed on isolated Pg-OMVs, which included phosphorylated dihydroceramide (PDHC) lipids and lipopolysaccharide (LPS), the latter in turn being contaminated with a highly pathogenic class of cysteine proteases, a key factor in neuronal cell degradation. Raman characterizations located lipopolysaccharide fingerprints in the vesicle structure and unveiled so far unproved aspects of the chemistry behind protein degradation induced by Pg-OMV contamination of SH-SY5Y cells. The observed alterations of cells' Raman profiles were then discussed in view of key factors including the formation of amyloid ß (Aß) plaques and hyperphosphorylated Tau neurofibrillary tangles, and the formation of cholesterol agglomerates that exacerbate AD pathologies.

Gut microbiome-derived glycine lipids are diet-dependent modulators of hepatic injury and atherosclerosis.

Oral and gut Bacteroidetes produce unique classes of serine-glycine lipodipeptides and glycine aminolipids that signal through host Toll-like receptor 2. These glycine lipids have also been detected in human arteries, but their effects on atherosclerosis are unknown. Here, we sought to investigate the bioactivity of bacterial glycine lipids in mouse models of atherosclerosis. Lipid 654 (L654), a serine-glycine lipodipeptide species, was first tested in a high-fat diet (HFD)-fed Ldlr-/- model of atherosclerosis. Intraperitoneal administration of L654 over 7 weeks to HFD-fed Ldlr-/- mice resulted in hypocholesterolemic effects and significantly attenuated the progression of atherosclerosis. We found that L654 also reduced liver inflammatory and extracellular matrix gene expression, which may be related to inhibition of macrophage activation as demonstrated in vivo by lower major histocompatibility complex class II gene expression and confirmed in cell experiments. In addition, L654 and other bacterial glycine lipids in feces, liver, and serum were markedly reduced alongside changes in Bacteroidetes relative abundance in HFD-fed mice. Finally, we tested the bioactivities of L654 and related lipid 567 in chow-fed Apoe-/- mice, which displayed much higher fecal glycine lipids relative to HFD-fed Ldlr-/- mice. Administration of L654 or lipid 567 for 7 weeks to these mice reduced the liver injury marker alanine aminotransferase, but other effects seen in Ldlr-/- were not observed. Therefore, we conclude that conditions in which gut microbiome-derived glycine lipids are lost, such as HFD, may exacerbate the development of atherosclerosis and liver injury, whereas correction of such depletion may protect from these disorders.

Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis.

Emerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal-cysteine-protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL-mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non-eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL-mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non-eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL-stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL-stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL-primed osteoclastogenesis was observed in male and female CatB-knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by P. gingivalis elevate RANKL-primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.

A novel phosphoglycerol serine-glycine lipodipeptide of Porphyromonas gingivalis is a TLR2 ligand.

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

Glycine Lipids of Porphyromonas gingivalis Are Agonists for Toll-Like Receptor 2.

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.

Systemic TLR2 tolerance enhances central nervous system remyelination.

BACKGROUND: Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by both inflammatory demyelination and impaired remyelination. Studies indicate that Toll-like receptor 2 (TLR2) signaling contributes to both the inflammatory component and the defective remyelination in MS. While most MS therapeutics target adaptive immunity, we recently reported that reducing TLR2 signaling in innate immune cells by inducing TLR2 tolerance attenuates adoptively transferred experimental autoimmune encephalomyelitis. Given that previous reports suggest TLR2 signaling also inhibits myelin repair, the objective of this study was to assess how reducing TLR2 signaling through TLR2 tolerance induction affects CNS myelin repair. METHODS: Chow containing 0.2% cuprizone was fed to male and female wild-type (WT) C57BL/6 mice or TLR2-deficient (TLR2-/-) mice for 5 weeks to induce demyelination. During a 2-week remyelination period following discontinuation of cuprizone, WT mice received either low dose TLR2 ligands to induce systemic TLR2 tolerance or vehicle control (VC). Remyelination was evaluated via electron microscopy and immunohistochemical analysis of microglia and oligodendrocytes in the corpus callosum. Statistical tests included 2-way ANOVA and Mann-Whitney U analyses. RESULTS: Inducing TLR2 tolerance in WT mice during remyelination significantly enhanced myelin recovery, restoring unmyelinated axon frequency and myelin thickness to baseline levels compared to VC-treated mice. Mechanistically, enhanced remyelination in TLR2 tolerized mice was associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. This result was confirmed in vitro by inducing TLR2 tolerance in WT microglia cultures. TLR2-/- mice, without TLR2 tolerance induction, also significantly enhanced myelin recovery compared to WT mice, adding confirmation that reduced TLR2 signaling is associated with enhanced remyelination. DISCUSSION: Our results suggest that reducing TLR2 signaling in vivo by inducing TLR2 tolerance significantly enhances myelin repair. Furthermore, the enhanced remyelination resulting from TLR2 tolerance induction is associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. While deletion of TLR2 would be an impractical approach in vivo, reducing innate immune signaling through TLR2 tolerance induction may represent a novel, two-pronged approach for treating both inflammatory and myelin repair components of MS.

Are Sphingolipids and Serine Dipeptide Lipids Underestimated Virulence Factors of Porphyromonas gingivalis?

The keystone periodontal pathogen Porphyromonas gingivalis produces phosphorylated dihydroceramide lipids (sphingolipids) such as phosphoethanolamine dihydroceramide (PE DHC) and phosphoglycerol dihydroceramide (PG DHC) lipids. Phosphorylated DHCs (PDHCs) from P. gingivalis can affect a number of mammalian cellular functions, such as potentiation of prostaglandin secretion from gingival fibroblasts, promotion of RANKL-induced osteoclastogenesis, promotion of apoptosis, and enhancement of autoimmunity. In P. gingivalis, these lipids affect anchoring of surface polysaccharides, resistance to oxidative stress, and presentation of surface polysaccharides (anionic polysaccharides and K-antigen capsule). In addition to phosphorylated dihydroceramide lipids, serine dipeptide lipids of P. gingivalis are implicated in alveolar bone loss in chronic periodontitis through interference with osteoblast differentiation and function and promotion of osteoclast activity. As a prerequisite for designation as bacterial virulence factors, bacterial sphingolipids and serine dipeptide lipids are recovered in gingival/periodontal tissues, tooth calculus, human blood, vascular tissues, and brain. In addition to P. gingivalis, other bacteria of the genera Bacteroides, Parabacteroides, Porphyromonas, Tannerella, and Prevotella produce sphingolipids and serine dipeptide lipids. The contribution of PDHCs and serine dipeptide lipids to the pathogenesis of periodontal and extraoral diseases may be an underappreciated area in microbe-host interaction and should be more intensively investigated.

TLR Tolerance as a Treatment for Central Nervous System Autoimmunity.

The role of TLR signaling in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is unclear. This role is especially controversial in models of adoptive transfer EAE in which no adjuvant and no TLR ligands are administered. We recently reported that a microbiome-derived TLR2 ligand, Lipid 654 (L654), is present in healthy human serum but significantly decreased in the serum of MS patients. This suggested that microbiome products that gain access to the systemic circulation, rather than being proinflammatory, may normally play an immune-regulatory role by maintaining a state of relative TLR tolerance. Therefore, a loss of microbiome-mediated TLR tolerance, as suggested by lower serum levels of L654, may play a role in the pathogenesis of MS. As proof of concept we asked whether administering low-level TLR2 ligands in adoptive transfer EAE induces TLR2 tolerance and attenuates disease. We administered low-level Pam2CSK4 or L654 to mice receiving encephalitogenic cells and in doing so induced both TLR2 tolerance and attenuation of EAE. Disease attenuation was accompanied in the CNS by a decrease in macrophage activation, a decrease in a specific proinflammatory macrophage population, and a decrease in Th17 cells. In addition, disease attenuation was associated with an increase in splenic type 1 regulatory T cells. Kinetic tolerance induction studies revealed a critical period for TLR2 involvement in adoptive transfer EAE. Overall, these results suggest that inducing TLR tolerance may offer a new approach to treating CNS autoimmune diseases such as MS.

Deposition and hydrolysis of serine dipeptide lipids of Bacteroidetes bacteria in human arteries: relationship to atherosclerosis.

Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls.

Simultaneous Determination of Absolute Configuration and Quantity of Lipopeptides Using Chiral Liquid Chromatography/Mass Spectrometry and Diastereomeric Internal Standards.

Lipopeptides promote innate immune response and are related to disease pathology. To investigate the newly emerging roles of lipopeptides, accurate measurements of stereoisomers with multiple chiral centers are essential yet challenging. This work uses (3R)- and (3S)-(15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, abbreviated as l-serine-(R+S)-Lipid 654, to develop a method that combines chiral liquid chromatography, a diastereomeric mixture of isotopically labeled internal standards, and multiple reaction monitoring mass spectrometry. The new method allows for simultaneously determining the absolute configuration and quantity of stereoisomers of bacteria-derived lipopeptides. Total lipid extracts of nine evaluated bacteria strains had different amounts, but only the (R)-isoform of l-serine-Lipid 654. The developed method also allowed for the first quantitative analysis of hydrolysis of a nonphospholipid as a novel substrate of honey bee venom phospholipase A2.

Phosphoglycerol dihydroceramide, a distinctive ceramide produced by Porphyromonas gingivalis, promotes RANKL-induced osteoclastogenesis by acting on non-muscle myosin II-A (Myh9), an osteoclast cell fusion regulatory factor.

Among several virulence factors produced by the periodontal pathogen Porphyromonas gingivalis (Pg), a recently identified novel class of dihydroceramide lipids that contains a long acyl-chain has the potential to play a pathogenic role in periodontitis because of its higher level of tissue penetration compared to other lipid classes produced by Pg. However, the possible impact of Pg ceramides on osteoclastogenesis is largely unknown. In the present study, we report that the phosphoglycerol dihydroceramide (PGDHC) isolated from Pg enhanced osteoclastogenesis in vitro and in vivo. Using RAW264.7 cells, in vitro assays indicated that PGDHC can promote RANKL-induced osteoclastogenesis by generating remarkably larger TRAP+ multinuclear osteoclasts compared to Pg LPS in a TLR2/4-independent manner. According to fluorescent confocal microscopy, co-localization of non-muscle myosin II-A (Myh9) and PGDHC was observed in the cytoplasm of osteoclasts, indicating the membrane-permeability of PGDHC. Loss- and gain-of-function assays using RNAi-based Myh9 gene silencing, as well as overexpression of the Myh9 gene, in RAW264.7 cells showed that interaction of PGDHC with Myh9 enhances RANKL-induced osteoclastogenesis. It was also demonstrated that PGDHC can upregulate the expression of dendritic cell-specific transmembrane protein (DC-STAMP), an important osteoclast fusogen, through signaling that involves Rac1, suggesting that interaction of PGDHC with Myh9 can elicit the cell signal that promotes osteoclast cell fusion. Taken together, our data indicated that PGDHC is a Pg-derived, cell-permeable ceramide that possesses a unique property of promoting osteoclastogenesis via interaction with Myh9 which, in turn, activates a Rac1/DC-STAMP pathway for upregulation of osteoclast cell fusion.

Convergent synthesis of a deuterium-labeled serine dipeptide lipid for analysis of biological samples.

Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, (3S)-l-serine] isolated from Porphyromonas gingivalis, in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria.

Effects of High-Pressure Treatment on Spores of Clostridium Species.

UNLABELLED: This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ∼150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at ≥400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods. IMPORTANCE: Spores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there is continued interest in ways to kill spores without compromising food quality. High hydrostatic pressure (HP) under appropriate conditions can inactivate bacterial spores. With growing use of HP for food pasteurization, advancement of HP for commercial production of sterile low-acid foods requires understanding of mechanisms of spores' interactions with HP. While much is known about HP germination and inactivation of spores of Bacillus species, how HP germinates and inactivates clostridial spores is less well understood. In this work we have tried to remedy this information deficit by examining germination of spores of Clostridium difficile and Clostridium perfringens by several HP and temperature levels. The results may give insight that could facilitate more efficient methods for spore eradication in food sterilization or pasteurization, biodecontamination, and health care.

Microbiome-derived bacterial lipids regulate gene expression of proinflammatory pathway inhibitors in systemic monocytes.

How the microbiome regulates responses of systemic innate immune cells is unclear. In the present study, our purpose was to document a novel mechanism by which the microbiome mediates crosstalk with the systemic innate immune system. We have identified a family of microbiome Bacteroidota-derived lipopeptides-the serine-glycine (S/G) lipids, which are TLR2 ligands, access the systemic circulation, and regulate proinflammatory responses of splenic monocytes. To document the role of these lipids in regulating systemic immunity, we used oral gavage with an antibiotic to decrease the production of these lipids and administered exogenously purified lipids to increase the systemic level of these lipids. We found that decreasing systemic S/G lipids by decreasing microbiome Bacteroidota significantly enhanced splenic monocyte proinflammatory responses. Replenishing systemic levels of S/G lipids via exogenous administration returned splenic monocyte responses to control levels. Transcriptomic analysis demonstrated that S/G lipids regulate monocyte proinflammatory responses at the level of gene expression of a small set of upstream inhibitors of TLR and NF-κB pathways that include Trem2 and Irf4. Consistent with enhancement in proinflammatory cytokine responses, decreasing S/G lipids lowered gene expression of specific pathway inhibitors. Replenishing S/G lipids normalized gene expression of these inhibitors. In conclusion, our results suggest that microbiome-derived S/G lipids normally establish a level of buffered signaling activation necessary for well-regulated innate immune responses in systemic monocytes. By regulating gene expression of inflammatory pathway inhibitors such as Trem2, S/G lipids merit broader investigation into the potential dysfunction of other innate immune cells, such as microglia, in diseases such as Alzheimer's disease.

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

Background: This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. Methods: We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. Results: We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. Conclusion: The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome.

The COVID-19 pandemic persists despite the availability of vaccines, and it is therefore crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of the oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, while PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed the modulatory activity of oral microbial communities varied among individuals in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19.