β-Neurexin Is a Ligand for the Staphylococcus aureus MSCRAMM SdrC

Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in β-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the β-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing β-neurexin on their surface. Taken together, our experimental results demonstrate that β-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N₂N₃ domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possible implications of this interaction are discussed.     

Azithromycin Reduces the Production of α-hemolysin and Biofilm Formation in Staphylococcus aureus

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases which are controlled by quorum sensing. In this study, we show that azithromycin was active against methicillin-resistant Staphylococcus aureus (MRSA) strains with MICs ranged from 32 to 64 μg/mL. Azithromycin at subinhibitory concentration, markedly reduced the production of α-hemolysin at (1/16MIC, 1/8MIC) and biofilm formation at (1/16MIC, 1/8MIC), respectively. The results indicated that sub-inhibitory concentrations of azithromycin decreased the production of α-hemolysin and biofilm formation in MRSA in a dose-dependent manner. Therefore, azithromycin may be useful in the treatment of α-hemolysin producing and biofilm formation MRSA infections.     

β-Lapachone activity in synergy with conventional antimicrobials against methicillin resistant Staphylococcus aureus strains

The aim of this study was to evaluate the antimicrobial activity of lapachol, α-lapachone, β-lapachone and six antimicrobials (ampicillin, amoxicillin/clavulanic acid, cefoxitin, gentamicin, ciprofloxacin and meropenem) against twelve strains of Staphylococcus aureus from which resistance phenotypes were previously determined by the disk diffusion method. Five S. aureus strains (LFBM 01, LFBM 26, LFBM 28, LFBM 31 and LFBM 33) showed resistance to all antimicrobial agents tested and were selected for the study of the interaction between β-lapachone and antimicrobial agents, busing checkerboard method. The criteria used to evaluate the synergistic activity were defined by the Fractional Inhibitory Concentration Index (FICI). Among the naphthoquinones, β-lapachone was the most effective against S. aureus strains. FICI values ranged from 0.07 to 0.5, suggesting a synergistic interaction against multidrug resistant S. aureus (MRSA) strains. An additive effect was observed with the combination β-lapachone/ciprofloxacin against the LFBM 33 strain. The combination of β-lapachone with cefoxitin showed no added benefit against LFBM 31 and LFBM 33 strains. This study demonstrated that, in general, β-lapachone combined with beta lactams antimicrobials, fluoroquinolones and carbapenems acts synergistically inhibiting MRSA strains.     

Caspase-1-Independent Interleukin-1β Is Required for Clearance of Bordetella pertussis Infections and Whole-Cell Vaccine-Mediated Immunity

Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1) response, which is required for the clearance of Bordetella pertussis. We show that IL-1β but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1β and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.     

Integrin-mediated mechanotransduction in IL-1β stimulated chondrocytes

Mechanical loading and interleukin-1β (IL-1β) influence the release of nitric oxide (^·NO) and prostaglandin E₂ (PGE₂) from articular chondrocytes via distinct signalling mechanisms. The exact nature of the interplay between the respective signalling pathways remains unclear. Recent studies have shown that integrins act as mechanoreceptors and may transduce extracellular stimuli into intracellular signals, thereby influencing cellular response. The current study demonstrates that the application of dynamic compression induced an inhibition of ^·NO and an upregulation of cell proliferation and proteoglycan synthesis in the presence and absence of IL-1β. PGE₂ release was not affected by dynamic compression in the absence of IL-1β but was inhibited in the presence of the cytokine. The integrin binding peptide, GRGDSP, abolished or reversed the compression-induced alterations in all four parameters assessed in the presence and absence of IL-1β. The non-binding control peptide, GRADSP, had no effect. These data clearly demonstrate that the metabolic response of the chondrocytes to dynamic compression in the presence and absence of IL-1β, are integrin mediated.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.     

Exercise Protects against Diet-Induced Insulin Resistance through Downregulation of Protein Kinase Cβ in Mice

Physical exercise is an important and effective therapy for diabetes. However, its underlying mechanism is not fully understood. Protein kinase Cβ (PKCβ) has been suggested to be involved in the pathogenesis of obesity and insulin resistance, but the role of PKCβ in exercise-induced improvements in insulin resistance is completely unknown. In this study, we evaluated the involvement of PKCβ in exercise-attenuated insulin resistance in high-fat diet (HFD)-fed mice. PKCβ^-/- and wild-type mice were fed a HFD with or without exercise training. PKC protein expression, body and tissue weight change, glucose and insulin tolerance, metabolic rate, mitochondria size and number, adipose inflammation, and AKT activation were determined to evaluate insulin sensitivity and metabolic changes after intervention. PKCβ expression decreased in both skeletal muscle and liver tissue after exercise. Exercise and PKCβ deficiency can alleviate HFD-induced insulin resistance, as evidenced by improved insulin tolerance. In addition, fat accumulation and mitochondrial dysfunction induced by HFD were also ameliorated by both exercise and PKCβ deficiency. On the other hand, exercise had little effect on PKCβ^-/- mice. Further, our data indicated improved activation of AKT, the downstream signal molecule of insulin, in skeletal muscle and liver of exercised mice, whereas PKCβ deficiency blunted the difference between sedentary and exercised mice. These results suggest that downregulation of PKCβ contributes to exercise-induced improvement of insulin resistance in HFD-fed mice.     

RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice

Fibroblasts are critical for wound contraction; a pivotal step in wound healing. They produce and modify the extracellular matrix (ECM) required for the proper tissue remodeling. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a key regulator of ECM homeostasis and turnover. However, its role in wound contraction is presently unknown. Here we describe that Transforming growth factor type β1 (TGF-β1), one of the main pro-fibrotic wound-healing promoting factors, decreases RECK expression in fibroblasts through the Smad and JNK dependent pathways. This TGF-β1 dependent downregulation of RECK occurs with the concomitant increase of β1-integrin, which is required for fibroblasts adhesion and wound contraction through the activation of focal adhesion kinase (FAK). Loss and gain RECK expression experiments performed in different types of fibroblasts indicate that RECK downregulation mediates TGF-β1 dependent β1-integrin expression. Also, reduced levels of RECK potentiate TGF-β1 effects over fibroblasts FAK-dependent contraction, without affecting its cognate signaling. The above results were confirmed on fibroblasts derived from the Reck ^+/- mice compared to wild type-derived fibroblasts. We observed that Reck ^+/- mice heal dermal wounds more efficiently than wild type mice. Our results reveal a critical role for RECK in skin wound contraction as a key mediator in the axis: TGF-β1—RECK- β1-integrin.     

Recombinant antimicrobial peptide hPAB-�� expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus

As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-??, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-?? products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9?Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of ??-factor by the STE13 protease. Expressed hPAB-?? was secreted into the culture medium at a level of 241.2?±?29.5?mg/L. Purified hPAB-?? with 95% homogeneity was obtained by 10?kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose? XL column. The two major protein species separated through a SOURCE? 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-?? with minimal inhibitory concentrations of 8?C64???g/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-??, and that expressed hPAB-?? in P. pastoris may be a potential antimicrobial agent against MRSA infection.     

Endogenous IL-1 in Cognitive Function and Anxiety: A Study in IL-1RI?/? Mice

Interleukin-1 (IL-1) is a key pro-inflammatory cytokine, produced predominantly by peripheral immune cells but also by glia and some neuronal populations within the brain. Its signalling is mediated via the binding of IL-1α or IL-1β to the interleukin-1 type one receptor (IL-1RI). IL-1 plays a key role in inflammation-induced sickness behaviour, resulting in depressed locomotor activity, decreased exploration, reduced food and water intake and acute cognitive deficits. Conversely, IL-1 has also been suggested to facilitate hippocampal-dependent learning and memory: IL-1RI^−/− mice have been reported to show deficits on tasks of visuospatial learning and memory. We sought to investigate whether there is a generalised hippocampal deficit in IL-1RI^−/− animals. Therefore, in the current study we compared wildtype (WT) mice to IL-1RI^−/− mice using a variety of hippocampal-dependent learning and memory tasks, as well as tests of anxiety and locomotor activity. We found no difference in performance of the IL-1RI^−/− mice compared to WT mice in a T-maze working memory task. In addition, the IL-1RI^−/− mice showed normal learning in various spatial reference memory tasks including the Y-maze and Morris mater maze, although there was a subtle deficit in choice behaviour in a spatial discrimination, beacon watermaze task. IL-1RI^−/− mice also showed normal memory for visuospatial context in the contextual fear conditioning paradigm. In the open field, IL-1RI^−/− mice showed a significant increase in distance travelled and rearing behaviour compared to the WT mice and in the elevated plus-maze spent more time in the open arms than did the WT animals. The data suggest that, contrary to prior studies, IL-1RI^−/− mice are not robustly impaired on hippocampal-dependent memory and learning but do display open field hyperactivity and decreased anxiety compared to WT mice. The results argue for a careful evaluation of the roles of endogenous IL-1 in hippocampal and limbic system function.     

ERRγ Is Not Required for Skeletal Development but Is a RUNX2-Dependent Negative Regulator of Postnatal Bone Formation in Male Mice

To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERRγ) deficiency on skeletal development and bone turnover, we utilized an ERRγ global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERRγ +/− males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERRγ +/− versus ERRγ +/+ stromal cell cultures, suggesting that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERRγ and RUNX2 interact in an ERRγ DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERRγ +/− but not ERRγ +/+ mice compared to their corresponding sense controls. Our data indicate that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner.