Topology and functional characterization of major outer membrane proteins of Treponema maltophilum and Treponema lecithinolyticum.

Numerous Treponema species are prevalent in the dysbiotic subgingival microbial community during periodontitis. The major outer sheath protein is a highly expressed virulence factor of the well-characterized species Treponema denticola. Msp forms an oligomeric membrane protein complex with adhesin and porin properties and contributes to host-microbial interaction. Treponema maltophilum and Treponema lecithinolyticum species are also prominent during periodontitis but are relatively understudied. Msp-like membrane surface proteins exist in T. maltophilum (MspA) and T. lecithinolyticum (MspTL), but limited information exists regarding their structural features or functionality. Protein profiling reveals numerous differences between these species, but minimal differences between strains of the same species. Using protein modeling tools, we predict MspA and MspTL monomeric forms to be large ß-barrel structures composed of 20 all-next-neighbor antiparallel ß strands which most likely adopt a homotrimer formation. Using cell fractionation, Triton X-114 phase partitioning, heat modifiability, and chemical and detergent release assays, we found evidence of amphiphilic integral membrane-associated oligomerization for both native MspA and MspTL in intact spirochetes. Proteinase K accessibility and immunofluorescence assays demonstrate surface exposure of MspA and MspTL. Functionally, purified recombinant MspA or MspTL monomer proteins can impair neutrophil chemotaxis. Expressions of MspA or MspTL with a PelB leader sequence in Escherichia coli also demonstrate surface exposure and can impair neutrophil chemotaxis in an in vivo air pouch model of inflammation. Collectively, our data demonstrate that MspA and MspTL membrane proteins can contribute to pathogenesis of these understudied oral spirochete species.

NRSF/REST lies at the intersection between epigenetic regulation, miRNA-mediated gene control and neurodevelopmental pathways associated with Intellectual disability (ID) and Schizophrenia.

Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association remain to be determined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a genetic disorder linked with neurodevelopmental disorders and associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA and leads to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Significantly, the EHMT1-regulated miRNA gene set not only controls NRSF/REST but is enriched for association for Intellectual Disability (ID) and schizophrenia. This reveals a broad molecular interaction between H3K9 demethylation, NSRF/REST regulation and risk for ID and Schizophrenia.

Mechanical characterization and adhesive properties of a dental adhesive modified with a polymer antibiotic conjugate.

This study is a follow up investigation on recent work by our group demonstrating synthesis, release and strong antibacterial character of resins modified with penicillin V (PV)-based polymer-antibiotic conjugates (PACs). Here, we aimed to evaluate the mechanical, bonding, and other relevant biomedical properties of a commercial adhesive resin modified with PV-PAC. Single Bond Plus (SB+) was modified with PAC containing 1.8 wt% conjugated PV. Adhesive resins were bonded to dentin from extracted human molars and restorative resin added. Beams of cross-sectional area of 0.9 ± 0.1 mm (Kutsch and Young, 2011) (n = 20) were obtained from the molars and tested for micro-tensile bond strength (µTBS) at 24 h and 4 months. For cohesive strength, hourglass beams (10 × 2 × 1 mm; n = 10) were assessed for ultimate tensile strength (UTS), beam-shaped specimens (25x2x2 mm; n = 10) evaluated for flexural strength and modulus (FS/FM) via three-point bending, and cylindrical specimens (3 × 2 mm; n = 10) assessed for ultimate compressive strength (UCS). For surface micro-hardness (MH), cylindrical specimens (3 × 2 mm; n = 6) were assessed before and after an EtOH challenge. The degree of conversion (DC) (5 × 1 mm; n = 6) was determined based on changes in absorbance ratio between peaks at ∼1637 cm-1 and ∼1608 cm-1 before and after curing of adhesive resins using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. To monitor water uptake and diffusion kinetics over a 28-day period, specimens (5 × 1mm) were desiccated, weighed and stored in deionized water. Control and PV-PAC modified adhesive resins demonstrated similar µTBS at 24 h and 4 months; both showing decrease in values after 4 months (p = 0.001 and 0.004). No significant differences between adhesive resins were shown in UTS, FS/FM or UCS (p<0.05). MH of PV-PAC adhesive resin was significantly reduced relative to the control (p<0.001). The DC values of the adhesive resins were not significantly different. While sorption and solubility were no different between materials, the diffusion coefficient of PV-PAC modified adhesive resin was higher than the control (p<0.001). We conclude that incorporation of PV-PAC with 1.8 wt% PV into an adhesive resin does not adversely affect its mechanical, bonding, and physical properties, thus providing a promising option for materials with long-term antibacterial character and on-demand release.

Copper@ZIF-8 Core-Shell Nanowires for Reusable Antimicrobial Face Masks.

SARS-CoV-2 and other respiratory viruses spread via aerosols generated by infected people. Face masks can limit transmission. However, widespread use of disposable masks consumes tremendous resources and generates waste. Here, a novel material for treating blown polypropylene filtration media used in medical-grade masks to impart antimicrobial activity is reported. To produce thin copper@ZIF-8 core-shell nanowires (Cu@ZIF-8 NWs), Cu NWs are stabilized using a pluronic F-127 block copolymer, followed by growth of ZIF-8 to obtain uniform core-shell structures. The Cu@ZIF-8 NWs are applied to filtration media by dip coating. Aerosol filtration efficiency decreases upon exposure to ethanol (solvent for dip-coating), but increases with addition of Cu@ZIF-8 NWs. Cu@ZIF-8 NWs shows enhanced antibacterial activity, compared to Cu NWs or ZIF-8 alone, against Streptococcus mutans and Escherichia coli. Antiviral activity against SARS-CoV-2 is assayed using virus-infected Vero E6 cells, demonstrating 55% inhibition of virus replication after 48 h by 1 µg of Cu@ZIF-8 NWs per well. Cu@ZIF-8 NWs' cytotoxicity is tested against four cell lines, and their effect on inflammatory response in A549 cells is examined, demonstrating good biocompatibility. This low-cost, scalable synthesis and straightforward deposition of Cu@ZIF-8 NWs onto filter media has great potential to reduce disease transmission, resource consumption, and environmental impact of waste.

Synthesis and antibacterial activity of polymer-antibiotic conjugates incorporated into a resin-based dental adhesive.

This work reports on polymer-antibiotic conjugates (PACs) as additives to resin-based restorative dental materials as a new strategy to convey sustained antibacterial character to these materials. Such antibacterial performance is expected to improve their longevity in the oral cavity. Using the previously reported ciprofloxacin (Cip)-based PAC as a control, a penicillin V (PV)-based PAC was investigated. The monomer-antibiotic conjugate (MAC) containing a methacrylate monomer group and a PV moiety was prepared via nucleophilic substitution between 2-chloroethyl methacrylate (CEMA) and penicillin V potassium (PVK). The PV-based PAC was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of the MAC with hydroxyethyl methacrylate (HEMA), and further characterized by 1H NMR and gel permeation chromatography (GPC) analysis. Antibiotic resistance was investigated by passaging bacteria in low concentrations of the antibiotic for 19 days, followed by a 48 h challenge at higher concentrations. Our results suggest that the development of antibiotic resistance is unlikely. Zone of inhibition (ZOI) assays revealed no clearing zones around PV-containing resins indicating minimal antibiotic leakage from the material. Similarly, MTT assay demonstrated that the antibiotic-containing specimens did not release cytotoxic byproducts that may inhibit human gingival fibroblast growth. Counting of colony-forming units in an S. mutans biofilm model was used to assess bacterial survival at baseline and after subjecting the antibiotic-containing resin specimens to an enzymatic challenge for 30 days. Significantly reduced bacterial counts were observed as the biofilm aged from 24 to 72 h, and salivary enzymatic exposure did not reduce the antibacterial efficacy of the discs, suggesting that PV-resin will be effective in reducing the re-incidence of dental caries.

Biocompatibility, mechanical, and bonding properties of a dental adhesive modified with antibacterial monomer and cross-linker.

OBJECTIVES: This study investigated the antibacterial, cytotoxicity, and mechanical properties of a dental adhesive modified with quaternary ammonium monomer ((2-acryloyloxyethyl)dimethyldodecylammonium bromide) and cross-linker (bis(2-acryloyloxyethyl)methyldodecylammonium bromide). MATERIALS AND METHODS: Monomer (M), cross-linker (C), or a combination of these (M + C) were incorporated into adhesive Adper Single Bond Plus (SB) in 5, 10, or 25% (as wt%). A colony-forming unit and MTT assays were used to evaluate antibacterial properties against Streptococcus mutans and cell viability. Resin-dentin beams (0.9 ± 0.1 mm2) were evaluated for micro-tensile bond strength (µTBS) after 24 h, 6 months, and 3 years. Hourglass specimens were evaluated for ultimate tensile strength (UTS) after 24 h, 1 week, and 6 months. Micro-hardness measurements after softening in ethanol were taken as an indirect assessment of the polymer cross-linking density. Kruskal-Wallis, one-way ANOVA, two-way ANOVA, and Student's t test were used for analysis of the antibacterial, cytotoxicity, µTBS, UTS, and hardness data, all with a significance level of p < 0.05. RESULTS: 10%M and 25%M demonstrated a significant reduction in S. mutans relative to SB (p < 0.001). No differences in cytotoxicity were detected for any of the groups. After 6 months, no changes in µTBS were shown for any of the groups. After 3 years, all groups evidenced a significant decrease in µTBS (p < 0.05) except 5%M, 5%C, and 5%M + 5%C. All groups demonstrated either stable or significantly increased UTS after 6 months. Except for the cross-linker groups, a significant decrease in micro-hardness was shown for all groups after softening in ethanol (p < 0.05). CONCLUSIONS: A 5-10% of monomer may render the resin antibacterial without a compromise to its mechanical and bonding properties. CLINICAL RELEVANCE: Biomodification of a resin adhesive with an antibacterial monomer and cross-linker may help improve the life span of adhesive restorations.

Treponema denticola stimulates Oncostatin M cytokine release and de novo synthesis in neutrophils and macrophages.

Oncostatin M (OSM) is a pleiotropic cytokine elevated in a number of inflammatory conditions including periodontal disease. OSM is produced by a variety of immune cells and has diverse functionality such as regulation of metabolic processes, cell differentiation, and the inflammatory response to bacterial pathogens. The oral cavity is under constant immune surveillance including complementary neutrophil and macrophage populations, due to a persistent symbiotic bacterial presence. Periodontal disease is characterized by a dysbiotic bacterial community, with an abundance of Treponema denticola. Despite strong associations with severe periodontal disease, the source and mechanism of the release of OSM have not been defined in the oral cavity. We show that OSM protein is elevated in the gingival epithelium and immune cell infiltrate during periodontal disease. Furthermore, salivary and oral neutrophil OSM is elevated in correlation with the presence of T. denticola. In an air pouch infection model, T. denticola stimulated higher levels of OSM than the oral pathogen Porphorymonas gingivalis, despite differential recruitment of innate immune cells suggesting T. denticola has distinct properties to elevate OSM levels. OSM release and transcription were increased in isolated human blood, oral neutrophils, or macrophages exposed to T. denticola in vitro as measured by ELISA, qPCR, and microscopy. Using transcription, translation, and actin polymerization inhibition, we found that T. denticola stimulates both OSM release through degranulation and de novo synthesis in neutrophils and also OSM release and synthesis in macrophages. Differential induction of OSM by T. denticola may promote clinical periodontal disease.

The Msp Protein of Treponema denticola Interrupts Activity of Phosphoinositide Processing in Neutrophils.

Periodontal disease is a significant health burden, causing tooth loss and poor oral and overall systemic health. Dysbiosis of the oral biofilm and a dysfunctional immune response drive chronic inflammation, causing destruction of soft tissue and alveolar bone supporting the teeth. Treponema denticola, a spirochete abundant in the plaque biofilm of patients with severe periodontal disease, perturbs neutrophil function by modulating appropriate phosphoinositide (PIP) signaling. Through a series of immunoblotting and quantitative PCR (qPCR) experiments, we show that Msp does not alter the gene transcription or protein content of key enzymes responsible for PIP3 signaling: 3' phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), or 5' Src homology 2 domain-containing inositol phosphatase 1 (SHIP1). Instead, using immunoblotting and enzyme-linked immunosorbent assays (ELISAs), we found that Msp activates PTEN through dephosphorylation specifically at the S380 site. Msp in intact organisms or outer membrane vesicles also restricts PIP signaling. SHIP1 phosphatase release was assessed using chemical inhibition and immunoprecipitation to show that Msp moderately decreases SHIP1 activity. Msp also prevents secondary activation of the PTEN/PI3K response. We speculate that this result is due to the redirection of the PIP3 substrate away from SHIP1 to PTEN. Immunofluorescence microscopy revealed a redistribution of PTEN from the cytoplasm to the plasma membrane following exposure to Msp, which may contribute to PTEN activation. Mechanisms of how T. denticola modulates and evades the host immune response are still poorly described, and here we provide further mechanistic evidence of how spirochetes modify PIP signaling to dampen neutrophil function. Understanding how oral bacteria evade the immune response to perpetuate the cycle of inflammation and infection is critical for combating periodontal disease to improve overall health outcomes.

Regulator of G protein signaling 12 enhances osteoclastogenesis by suppressing Nrf2-dependent antioxidant proteins to promote the generation of reactive oxygen species.

Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation. We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted Rgs12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed Rgs12 in RAW264.7 cells. Rgs12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone, decreased osteoclast number and activity but no change in osteoblast number and bone formation. Rgs12 overexpression increased osteoclast number and size, and bone resorption activity. Proteomics analysis of Rgs12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2, the master regulator of oxidative stress. We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in Rgs12-deficient cells. Conversely, Rgs12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome, and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB, which was abrogated by antioxidant treatment. Our study therefore identified a novel role of Rgs12 in regulating Nrf2, thereby controlling cellular redox state and osteoclast differentiation.

Polymer-antibiotic conjugates as antibacterial additives in dental resins.

Affecting the vast majority of human beings, dental caries is a premier concern of worldwide dental health. As the most commonly used restorative material to treat dental caries, resin-based composites (RBCs) lack antibacterial properties leading to quite limited restoration lifetimes. The objective of this study is to develop a polymer-antibiotic conjugate (PAC) as an effective antibacterial additive for RBCs. A monomer-antibiotic conjugate (MAC) with significant solubility was prepared by an esterification reaction of tert-butyloxycarbonyl (Boc)-protected ciprofloxacin (Cip) and 2-hydroxyethyl methacrylate (HEMA). The Cip-containing PAC with well-controlled molecular weight and composition was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization of the MAC with HEMA (1 : 3 molar ratio), followed by the removal of Boc from the resulting copolymer. The antibacterial dental resin was then prepared by incorporating the PAC into a commercial resin, and their properties and antibacterial performance against Streptococcus mutans were tested. In vitro experiments revealed a very slow release of Cip, which resulted in significant killing effectiveness against Streptococcus mutans nonetheless, as observed through zone of inhibition assessment and SEM imaging. The promising antibacterial properties of these resins indicate that incorporating a PAC as an additive is a valid strategy to generate antibacterial materials for dental applications.

Expression of the Oligopeptide Permease Operon of Moraxella catarrhalis Is Regulated by Temperature and Nutrient Availability.

Moraxella catarrhalis causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Together, these two conditions contribute to enormous morbidity and mortality worldwide. The oligopeptide permease (opp) ABC transport system is a nutritional virulence factor important for the utilization of peptides. The substrate binding protein OppA, which binds peptides for uptake, is a potential vaccine antigen, but little was known about the regulation of gene expression. The five opp genes oppB, oppC, oppD, oppF, and oppA are in the same open reading frame. Sequence analysis predicted two promoters, one located upstream of oppB and one within the intergenic region between oppF and oppA. We have characterized the gene cluster as an operon with two functional promoters and show that cold shock at 26°C for ≤ 0.5 h and the presence of a peptide substrate increase gene transcript levels. Additionally, the putative promoter upstream of oppA contributes to the transcription of oppA but is not influenced by the same environmental cues as the promoter upstream of oppB. We conclude that temperature and nutrient availability contribute to the regulation of the Opp system, which is an important nutritional virulence factor in M. catarrhalis.

Expression of IgA Proteases by Haemophilus influenzae in the Respiratory Tract of Adults With Chronic Obstructive Pulmonary Disease.

BACKGROUND: Immunoglobulin (Ig)A proteases of Haemophilus influenzae are highly specific endopeptidases that cleave the hinge region of human IgA1 and also mediate invasion and trafficking in human respiratory epithelial cells, facilitating persistence of H. influenzae. Little is known about the expression of IgA proteases in clinical settings of H. influenzae infection. METHODS: We identified and characterized IgA protease genes in H. influenzae and studied their expression and proteolytic specificity, in vitro and in vivo in 169 independent strains of H. influenzae collected longitudinally over 10 years from adults with chronic obstructive pulmonary disease. RESULTS: The H. influenzae pangenome has 2 alleles of IgA protease genes; all strains have igaA, and 40% of strains have igaB. Each allele has 2 variants with differing proteolytic specificities for human IgA1. A total of 88% of 169 strains express IgA protease activity. Expression of the 4 forms of IgA protease varies among strains. Based on the presence of IgA1 fragments in sputum samples, each of the different forms of IgA protease is selectively expressed in the human airways during infection. CONCLUSIONS: Four variants of IgA proteases are variably expressed by H. influenzae during infection of the human airways.

Role of the oligopeptide permease ABC Transporter of Moraxella catarrhalis in nutrient acquisition and persistence in the respiratory tract.

Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.

Substrate binding protein SBP2 of a putative ABC transporter as a novel vaccine antigen of Moraxella catarrhalis.

Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae, M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis. Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-µg and 50-µg doses (P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis.