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Alzheimer’s disease (AD) is characterized by the accumulation of amyloid beta plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. This study computationally investigated natural neem compounds (limonoids) and gut microbiome metabolites for their inhibitory potential against key AD targets. Molecular docking analyses were performed on approximately 200 neem phytochemicals and 9 microbial metabolites against beta-secretase 1 (BACE1), gingipain cysteine protease, and tau oligomerization receptors using AutoDock. BBB permeability was computationally evaluated using six molecular descriptors: molecular weight, LogP, hydrogen bond acceptors/donors, polar surface area, and rotatable bonds, categorizing compounds as highly or poorly BBB permeable based on established predictive criteria. The results revealed superior binding affinities of limonoids, notably Rutin (− 9.642 kcal/mol), 7-benzoylnimbocinol (− 9.706 kcal/mol), and tirucallol (− 9.488 kcal/mol) against BACE1, gingipain protease, and tau oligomerization receptors, respectively. These compounds exhibited key interactions through hydrogen bonding with Gly34, Asn233 (rutin-BACE1), Lys311, and Asn363 (7-benzoylnimbocinol-gingipain) and hydrophobic interactions with Ile40 and Ile48 (tirucallol-tau). While these limonoids demonstrated binding affinities exceeding melatonin by > 30%, their BBB permeability profiles necessitate sophisticated delivery strategies. Among gut microbiome metabolites, melatonin showed consistent binding across all targets (− 7.079 to − 8.452 kcal/mol). These findings establish limonoids’ superiority over gut microbiome metabolites and highlight their therapeutic potential as multi-target inhibitors in AD pathology, warranting investment in nanocarrier systems for optimizing BBB penetration.Article HighlightsIn silico, screening identified neem phytochemicals as potent inhibitors of key Alzheimer's disease targets, exhibiting superior binding affinities compared to gut microbiome metabolites.Compounds like rutin, nimbolin B, and protolimonoids demonstrated remarkable docking scores against the BACE1 enzyme, a crucial therapeutic target in the amyloidogenic pathway.Indole melatonin consistently displays robust interactions across BACE1, gingipain protease, and tau protein receptors, highlighting its potential as a privileged scaffold for Alzheimer’s drug development.

Among oral tissues, the periodontium is permanently subjected to mechanical forces resulting from chewing, mastication, or orthodontic appliances. Molecularly, these movements induce a series of subsequent signaling processes, which are embedded in the biological concept of cellular mechanotransduction (MT). Cell and tissue structures, ranging from the extracellular matrix (ECM) to the plasma membrane, the cytosol and the nucleus, are involved in MT. Dysregulation of the diverse, fine-tuned interaction of molecular players responsible for transmitting biophysical environmental information into the cell’s inner milieu can lead to and promote serious diseases, such as periodontitis or oral squamous cell carcinoma (OSCC). Therefore, periodontal integrity and regeneration is highly dependent on the proper integration and regulation of mechanobiological signals in the context of cell behavior. Recent experimental findings have increased the understanding of classical cellular mechanosensing mechanisms by both integrating exogenic factors such as bacterial gingipain proteases and newly discovered cell-inherent functions of mechanoresponsive co-transcriptional regulators such as the Yes-associated protein 1 (YAP1) or the nuclear cytoskeleton. Regarding periodontal MT research, this review offers insights into the current trends and open aspects. Concerning oral regenerative medicine or weakening of periodontal tissue diseases, perspectives on future applications of mechanobiological principles are discussed.

Porphyromonas gingivalis is a member of the dysbiotic oral microbiome and a “keystone pathogen” that causes severe periodontal disease, which is among the most prevalent infectious diseases. Part of the virulence factors secreted by P. gingivalis are the essential cysteine peptidases gingipain K (Kgp) and R (RgpA and RgpB), which account for 85% of the extracellular proteolytic activity of the pathogen and are thus prime targets for inhibition. We report the high-resolution (1.20 Å) complex structure of Kgp with KYT-36, a peptide-derived, potent, bioavailable and highly selective inhibitor, which is widely used for studies in vitro , in cells and in vivo . Sub-nanomolar inhibition of Kgp is achieved by tight binding to the active-site cleft, which is covered for its sub-sites S 3 through S 1 ’ under establishment of nine hydrophobic interactions, 14 hydrogen bonds and one salt bridge. In addition, an inhibitor carbonyl carbon that mimics the scissile carbonyl of substrates is pyramidalized and just 2.02 Å away from the catalytic nucleophile of Kgp, C 477 Sγ. Thus, the crystal structure emulates a reaction intermediate of the first nucleophilic attack during catalysis of cysteine peptidases. The present study sets the pace for the development of tailored next-generation drugs to tackle P. gingivalis .

Periodontal pathogen Porphyromonas gingivalis ( P. gingivalis ) is believed to possess immune evasion capabilities, but it remains unclear whether this immune evasion is related to host gene alternative splicing (AS). In this study, RNA-sequencing revealed significant changes in both AS landscape and transcriptomic profile of macrophages following P. gingivalis infection with/without knockout of gingipain (a unique toxic protease of P. gingivalis ). P. gingivalis infection increased the PD-L1 transcripts expression and selectively upregulated a specific coding isoform that more effectively binds to PD-1 on T cells, thereby inhibiting immune function. Biological experiments also detected AS switch of PD-L1 in P. gingivalis -infected or gingipain-treated macrophages. AlphaFold 3 predictions indicated that the protein docking compatibility between PD-1 and P. gingivalis -upregulated PD-L1 isoform was over 80% higher than another coding isoform. These findings suggest that P. gingivalis employs gingipain to modulate the AS of PD-L1, facilitating immune evasion.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

Periodontitis (PD) is a chronic inflammatory disease of the gingiva, with a high prevalence. Clinical reports indicate the significant role of PD in the development of comorbidities, including Herpesviridae infections; however, the molecular basis of this phenomenon has not yet been described. In our work, we uncovered a novel molecular mechanism by which the interferon-dependent antiviral response is tailored by the cysteine protease of P. gingivalis —Kgp. Using gingival keratinocytes and a model of human gingiva, we have demonstrated that lysin-specific gingipain attenuates the antiviral response and promotes the propagation of herpes simplex virus-1, which is one of the most frequently identified viruses in patients suffering from PD. These findings expand our knowledge of the mechanisms underlying polymicrobial infections and may provide a basis for considering PD as a gateway to viral infection.

Porphyromonas gingivalis (Pg) is a keystone pathogen in periodontitis, a highly prevalent disease manifested by chronic inflammation of the periodontium, alveolar bone resorption and tooth loss. During periodontitis pathobionts such as Pg can enter the bloodstream and growing evidence correlates periodontitis with increased risk of cardiovascular and neurodegenerative diseases. However, the mechanism by which immune cells respond to Pg challenge in vivo remains elusive. Pg produce aggressive proteolytic virulence factors termed gingipains which not only provide nutrients necessary for bacterial growth, but also subvert the host immune response, facilitating bacterial survival. Using transgenic zebrafish with fluorescently labelled macrophages and neutrophils, the role of gingipains in bacterial survival and interaction with phagocytes during systemic and local infection was examined. In contrast to the wild-type (W83) Pg , isogenic gingipain-null ( ΔK/R-ab) or wild-type Pg treated with gingipain inhibitors caused less zebrafish mortality, bacteria were rapidly phagocytosed, acidified in phagosomes and eradicated when systemically injected, showing that gingipains are instrumental in preventing phagocytosis and intracellular killing of Pg by innate immune cells. Moreover, Pg were predominantly phagocytosed by macrophages, and gingipain depletion/inactivation increased bacterial phagocytosis when bacteria were injected either systemically or locally in the otic vesicle, with less bacteria internalised by neutrophils. This phenomenon was Pg -specific as Fusobacterium nucleatum caused neutrophil recruitment that then effectively phagocytosed these bacteria. These data demonstrate the important role of phagocytes, especially macrophages, in combating Pg infection and highlight the crucial protective role of gingipains in subverting the innate immune response. This study also emphasizes the advantages of using zebrafish to study interactions of Pg with phagocytes in vivo in real-time, providing a valuable experimental system for testing new therapeutic strategies aimed at reducing periodontal-associated systemic or neurodegenerative disease.

Porphyromonas gingivalis, a major pathogen in periodontitis, produces KGP (Lys-gingipain), a cysteine protease that enhances bacterial virulence by promoting tissue invasion and immune evasion. Recent studies highlight microRNAs' role in viral latency, potentially affecting lytic replication through host mechanisms. Herpes virus (HSV) establishes latency via interactions between microRNA-6 (miRH-6) and the ICP4 transcription factor in neural ganglia. This suggests a potential link between periodontitis and HSV-induced latency. This study aims to identify and validate the insilico inhibitory interaction of P. gingivalis KGP with ICP4 transcripts and correlate the presence of viral latency-associated transcript micro-RNA-6 with periodontitis. Computational docking analysis was performed to investigate the potential interaction between ICP4 and KGP gingipain. The binding energy and RMSD ligand values were calculated to determine the interaction's strength. Ten patients with recurrent clinical attachment loss despite conventional therapy were included in the clinical study. Subgingival tissue samples were collected post-phase I therapy, and HSV microRNA-6 presence was detected via polymerase chain reaction and confirmed through gel electrophoresis. Computational docking identified the ICP4-KGP gingipain complex with the lowest binding energy (-288.29 kJ mol^1) and an RMSD ligand of 1.5 Angstroms, indicating strong interaction potential. Gel electrophoresis confirmed miRH-6 presence in all samples. The identification of miRNA-6 in periodontitis patients and the strong interaction potential between P. gingivalis KGP gingipain and ICP4 transcripts indicate a possible link between bacterial virulence factors and viral latency dynamics in periodontal tissues. These results highlight the complex interplay between oral pathogens, viral microRNAs, and host immune responses in periodontitis.

Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains—cysteine proteases from Porphyromonas gingivalis —are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis , based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes—cells at the forefront of the fight against bacteria—cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.

Porphyromonas gingivalis , a bacterium associated with periodontal disease, is a suspected cause of Alzheimer’s disease. This bacterium is reliant on gingipain proteases, which cleave host proteins after arginine and lysine residues. To characterize gingipain susceptibility, we performed enrichment analyses of arginine and lysine proportion proteome-wide. Genes differentially expressed in brain samples with detected P. gingivalis reads were also examined. Genes from these analyses were tested for functional enrichment and specific neuroanatomical expression patterns. Proteins in the SRP-dependent cotranslational protein targeting to membrane pathway were enriched for these residues and previously associated with periodontal and Alzheimer’s disease. These ribosomal genes are up-regulated in prefrontal cortex samples with detected P. gingivalis sequences. Other differentially expressed genes have been previously associated with dementia ( ITM2B , MAPT , ZNF267 , and DHX37 ). For an anatomical perspective, we characterized the expression of the P. gingivalis associated genes in the mouse and human brain. This analysis highlighted the hypothalamus, cholinergic neurons, and the basal forebrain. Our results suggest markers of neural P. gingivalis infection and link the cholinergic and gingipain hypotheses of Alzheimer’s disease.