Type I interferon regulation by USP18 is a key vulnerability in cancer.

Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.

GP96 Drives Exacerbation of Secondary Bacterial Pneumonia following Influenza A Virus Infection.

Influenza A virus (IAV) infection predisposes the host to secondary bacterial pneumonia, known as a major cause of morbidity and mortality during influenza virus epidemics. Analysis of interactions between IAV-infected human epithelial cells and Streptococcus pneumoniae revealed that infected cells ectopically exhibited the endoplasmic reticulum chaperone glycoprotein 96 (GP96) on the surface. Importantly, efficient pneumococcal adherence to epithelial cells was imparted by interactions with extracellular GP96 and integrin αV, with the surface expression mediated by GP96 chaperone activity. Furthermore, abrogation of adherence was gained by chemical inhibition or genetic knockout of GP96 as well as addition of RGD peptide, an inhibitor of integrin-ligand interactions. Direct binding of extracellular GP96 and pneumococci was shown to be mediated by pneumococcal oligopeptide permease components. Additionally, IAV infection induced activation of calpains and Snail1, which are responsible for degradation and transcriptional repression of junctional proteins in the host, respectively, indicating increased bacterial translocation across the epithelial barrier. Notably, treatment of IAV-infected mice with the GP96 inhibitor enhanced pneumococcal clearance from lung tissues and ameliorated lung pathology. Taken together, the present findings indicate a viral-bacterial synergy in relation to disease progression and suggest a paradigm for developing novel therapeutic strategies tailored to inhibit pneumococcal colonization in an IAV-infected respiratory tract. IMPORTANCE Secondary bacterial pneumonia following an influenza A virus (IAV) infection is a major cause of morbidity and mortality. Although it is generally accepted that preceding IAV infection leads to increased susceptibility to secondary bacterial infection, details regarding the pathogenic mechanism during the early stage of superinfection remain elusive. Here, we focused on the interaction of IAV-infected cells and Streptococcus pneumoniae, which revealed that human epithelial cells infected with IAV exhibit a cell surface display of GP96, an endoplasmic reticulum chaperon. Notably, extracellular GP96 was shown to impart efficient adherence for secondary infection by S. pneumoniae, and GP96 inhibition ameliorated lung pathology of superinfected mice, indicating it to be a useful target for development of therapeutic strategies for patients with superinfection.

Streptococcus pneumoniae Surface Adhesin PfbA Exhibits Host Specificity by Binding to Human Serum Albumin but Not Bovine, Rabbit and Porcine Serum Albumins.

PfbA (Plasmin(ogen) and Fibronectin Binding protein A) is an adhesin present on the surface of Streptococcus pneumoniae. Initial studies characterized PfbA as plasmin(ogen) and fibronectin binding protein and later it was found that it binds with many other proteins of the extracellular matrix such as fibrinogen, collagen and laminin. It also binds to blood protein human serum albumin (HSA). Interestingly, PfbA exhibits no binding with serum albumins of bovine (BSA), rabbit (RSA) and porcine (PSA) which are sequentially and structurally homologous to HSA. This suggests that PfbA is likely involved in host specificity. Therefore, to get more insights into this aspect, a detailed analysis, which includes the interaction of rPfbA with HSA/BSA/RSA/PSA at different pHs by bio-layer interferometry, comparison of sequences and surface electrostatic potential of HSA/BSA/RSA/PSA, lysine modification of HSA by succinylation and subsequent interaction analysis of succinylated HSA with rPfbA and the secondary structural content estimation by FT-IR spectroscopy was carried out. Since large protrusions are another important geometric feature of protein surfaces, the property was also analyzed for HSA/BSA/RSA/PSA. The results of the above studies clearly suggest that the rPfbA exhibits host specificity by selectively binding only to HSA and not with its homologous BSA/RSA/PSA. Since the three dimensional structures of these albumins are highly similar, it is likely that rPfbA utilizes the differences in the surface electrostatic charge in combination with surface protrusions of HSA/BSA/RSA/PSA for the selective molecular recognition process and this feature may be important in the pathogenesis of pneumococcal infection.

Streptococcus pyogenes Transcriptome Changes in the Inflammatory Environment of Necrotizing Fasciitis.

Streptococcus pyogenes is a major cause of necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection. At the host infection site, the local environment and interactions between the host and bacteria have effects on bacterial gene expression profiles, while the gene expression pattern of S. pyogenes related to this disease remains unknown. In this study, we used a mouse model of necrotizing fasciitis and performed RNA-sequencing (RNA-seq) analysis of S. pyogenes M1T1 strain 5448 by isolating total RNA from infected hind limbs obtained at 24, 48, and 96 h postinfection. RNA-seq analysis results identified 483 bacterial genes whose expression was consistently altered in the infected hindlimbs compared to their expression under in vitro conditions. Genes showing consistent enrichment during infection included 306 encoding molecules involved in virulence, carbohydrate utilization, amino acid metabolism, trace-metal transport, and the vacuolar ATPase transport system. Surprisingly, drastic upregulation of 3 genes, encoding streptolysin S precursor (sagA), cysteine protease (speB), and secreted DNase (spd), was noted in the present mouse model (log2 fold change, >6.0, >9.4, and >7.1, respectively). Conversely, the number of consistently downregulated genes was 177, including those associated with the oxidative stress response and cell division. These results suggest that in necrotizing fasciitis, S. pyogenes shows an altered metabolism, decreased cell proliferation, and upregulation of expression of major toxins. Our findings are considered to provide critical information for developing novel treatment strategies and vaccines for necrotizing fasciitis.IMPORTANCE Necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection, is principally caused by S. pyogenes The inflammatory environment at the site of infection causes global gene expression changes for survival of the bacterium and pathogenesis. However, no known study regarding transcriptomic profiling of S. pyogenes in cases of necrotizing fasciitis has been presented. We identified 483 bacterial genes whose expression was consistently altered during infection. Our results showed that S. pyogenes infection induces drastic upregulation of the expression of virulence-associated genes and shifts metabolic pathway usage. In particular, high-level expression of toxins, such as cytolysins, proteases, and nucleases, was observed at infection sites. In addition, genes identified as consistently enriched included those related to metabolism of arginine and histidine as well as carbohydrate uptake and utilization. Conversely, genes associated with the oxidative stress response and cell division were consistently downregulated during infection. The present findings provide useful information for establishing novel treatment strategies.

Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA.

Streptococcus pneumoniae is a Gram-positive bacterium belonging to the oral streptococcus species, mitis group. This pathogen is a leading cause of community-acquired pneumonia, which often evades host immunity and causes systemic diseases, such as sepsis and meningitis. Previously, we reported that PfbA is a ß-helical cell surface protein contributing to pneumococcal adhesion to and invasion of human epithelial cells in addition to its survival in blood. In the present study, we investigated the role of PfbA in pneumococcal pathogenesis. Phylogenetic analysis indicated that the pfbA gene is highly conserved in S. pneumoniae and Streptococcus pseudopneumoniae within the mitis group. Our in vitro assays showed that PfbA inhibits neutrophil phagocytosis, leading to pneumococcal survival. We found that PfbA activates NF-κB through TLR2, but not TLR4. In addition, TLR2/4 inhibitor peptide treatment of neutrophils enhanced the survival of the S. pneumoniae ΔpfbA strain as compared to a control peptide treatment, whereas the treatment did not affect survival of a wild-type strain. In a mouse pneumonia model, the host mortality and level of TNF-α in bronchoalveolar lavage fluid were comparable between wild-type and ΔpfbA-infected mice, while deletion of pfbA decreased the bacterial burden in bronchoalveolar lavage fluid. In a mouse sepsis model, the ΔpfbA strain demonstrated significantly increased host mortality and TNF-α levels in plasma, but showed reduced bacterial burden in lung and liver. These results indicate that PfbA may contribute to the success of S. pneumoniae species by inhibiting host cell phagocytosis, excess inflammation, and mortality by interacting with TLR2.

Streptococcus pyogenes CAMP factor promotes calcium ion uptake in RAW264.7 cells.

Streptococcus pyogenes is a bacterium that causes systemic diseases such as pharyngitis and toxic shock syndrome. S. pyogenes produces molecules that inhibit the function of the human immune system, thus allowing growth and spread of the pathogen in tissues. It is known that S. pyogenes CAMP factor induces vacuolation in macrophages; however, the mechanism remains unclear. In the current study, the mechanism by which CAMP factor induces vacuolation in macrophages was investigated. CAMP factor was found to induce calcium ion uptake in murine macrophage RAW264.7 cells. In addition, EDTA inhibited calcium ion uptake and vacuolation in the cells. The L-type voltage-dependent calcium ion channel blockers nifedipine and verapamil reduced vacuolation. Furthermore, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin also inhibited the vacuolation induced by CAMP factor. Fluorescent microscopy revealed that clathrin localized to the vacuoles. These results suggest that the vacuolation is related to calcium ion uptake by RAW264.7 cells via L-type voltage-dependent calcium ion channels. Therefore, it was concluded that the vacuoles induced by S. pyogenes CAMP factor in macrophages are clathrin-dependent endosomes induced by activation of the phosphoinositide 3-kinase signaling pathway through calcium ion uptake.

Streptococcal Cysteine Protease-Mediated Cleavage of Desmogleins Is Involved in the Pathogenesis of Cutaneous Infection.

Streptococcus pyogenes is responsible for a wide variety of cutaneous infections ranging from superficial impetigo to fulminant invasive necrotizing fasciitis. Dysfunction of desmosomes is associated with the pathogenesis of cutaneous diseases. We identified streptococcal pyrogenic exotoxin B (SpeB) as a proteolytic factor that cleaves the extracellular domains of desmoglein 1 and 3. In an epicutaneous infection model, lesional skin infected with an speB deletion mutant were significantly smaller as compared to those caused by the wild-type strain. Furthermore, immunohistological analysis indicated cleavage of desmogleins that developed around the invasion site of the wild-type strain. In contrast, the speB mutant was preferentially found on the epidermis surface layer. Taken together, our findings provide evidence that SpeB-mediated degradation of desmosomes has a pathogenic role in development of S. pyogenes cutaneous infection.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

Atg7 Activates an Autophagy-Essential Ubiquitin-like Protein Atg8 through Multi-Step Recognition.

Atg8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for Atg8, and it activates the C-terminal Gly116 of Atg8 using ATP. Here, we report the crystal structure of Atg8 bound to the C-terminal domain of Atg7 in an unprecedented mode. Atg8 neither contacts with the central ß-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7-Atg8 structures. Instead, Atg8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes Atg8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction.

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway.

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine-threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells.

Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice.

The Streptococcus pyogenes phospholipase A2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes, which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the ΔslaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.

Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for local suppurative and life-threatening invasive systemic diseases. Interaction of GAS with human plasminogen (PLG) is a salient characteristic for promoting their systemic dissemination. In the present study, a serotype M28 strain was found predominantly localized in tricellular tight junctions of epithelial cells cultured in the presence of PLG. Several lines of evidence indicated that interaction of PLG with tricellulin, a major component of tricellular tight junctions, is crucial for bacterial localization. A site-directed mutagenesis approach revealed that lysine residues at positions 217 and 252 within the extracellular loop of tricellulin play important roles in PLG-binding activity. Additionally, we demonstrated that PLG functions as a molecular bridge between tricellulin and streptococcal surface enolase (SEN). The wild type strain efficiently translocated across the epithelial monolayer, accompanied by cleavage of transmembrane junctional proteins. In contrast, amino acid substitutions in the PLG-binding motif of SEN markedly compromised those activities. Notably, the interaction of PLG with SEN was dependent on PLG species specificity, which influenced the efficiency of bacterial penetration. Our findings provide insight into the mechanism by which GAS exploits host PLG for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions.

Injection of Dental Pulp Stem Cells Promotes Healing of Damaged Bladder Tissue in a Rat Model of Chemically Induced Cystitis.

Dental pulp stem cells (DPSCs) are reported as sources of mesenchymal stem cells (MSCs). MSCs are used as cell therapy options for various diseases. The present study examined the healing effects of DPSC injection on damaged bladder tissue in a chemically induced cystitis rat model. Cystitis was induced by hydrochloride injection into the bladder of female F344/NSlc rats. On the following day, DPSCs suspended in phosphate-buffered saline (PBS) were injected into the bladder and maintained for 1 h (DPSC injection group), while PBS alone was injected as the standard for comparison (PBS injection group). After 2 days following injection, considerable submucosal edema, vascular structure destruction, hemorrhage, and inflammatory cell invasion were observed both in the DPSC and PBS injection groups, with no difference in their degree of submucosal edema and hemorrhage. Six days after injection, vascular structure regeneration was observed in both groups; however, unlike the DPSC injection group, the PBS injection group showed traces of submucosal edema and hemorrhage. These results correlated with tissue concentrations of myeloperoxidase (MPO) and the inflammatory cytokines IL-1ß, IL-6, and TNF-α. Furthermore, the intercontraction interval was prolonged, and the frequency of nociceptive behaviors was reduced in the DPSC injection group compared with the PBS injection group. DPSCs were found on the bladder epithelium until day 3 after injection. In the DPSC-conditioned media (CM), the trophic factors FGF-2, VEGF, and the C-C and C-X-C families of chemokines were detected. The results of DPSC injection into the cystitis rat model suggested that the injected cells promote the healing of the damaged bladder tissue by exerting various trophic effects while localizing on the bladder epithelium and that MSC injection is a potential novel therapy for interstitial cystitis/painful bladder syndrome.

Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability.

The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogs are not bioavailable. To increase cellular permeability of mevalonate analogs, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogs by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogs in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates.

Endoplasmic reticulum protein BI-1 regulates Ca²âº-mediated bioenergetics to promote autophagy.

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²âº mediated by inositol triphosphate receptors (IP3Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP3R-dependent manner. By reducing steady-state levels of ER Ca²âº via IP3Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1's ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²âº signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.

α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia throughout the world, with high morbidity and mortality rates. A major feature of pneumococcal pneumonia is abundant neutrophil infiltration. In this study, we identified S. pneumoniae α-enolase as a neutrophil binding protein in ligand blot assay and mass spectrometry findings. Scanning electron microscopic and fluorescence microscopic analyses also revealed that S. pneumoniae α-enolase induces formation of neutrophil extracellular traps, which have been reported to bind and kill microbes. In addition, cytotoxic assay results showed that α-enolase dose-dependently increased the release of extracellular lactate dehydrogenase from human neutrophils as compared with untreated neutrophils. Furthermore, an in vitro cell migration assay using Chemotaxicell culture chambers demonstrated that α-enolase possesses neutrophil migrating activity. Interestingly, bactericidal assay findings showed that α-enolase increased neutrophil extracellular trap-dependent killing of S. pneumoniae in human blood. Moreover, pulldown assay and mass spectrometry results identified myoblast antigen 24.1D5 as an α-enolase-binding protein on human neutrophils, whereas flow cytometric analysis revealed that 24.1D5 was expressed on human neutrophils, but not on human monocytes or T cells. Together, our results indicate that α-enolase from S. pneumoniae increases neutrophil migrating activity and induces cell death of human neutrophils by releasing neutrophil extracellular traps. Furthermore, we found that myoblast antigen 24.1D5, which expressed on the surface of neutrophils, bound to α-enolase of S. pneumoniae.

S-carboxymethylcysteine inhibits adherence of Streptococcus pneumoniae to human alveolar epithelial cells.

Streptococcus pneumoniae is a major pathogen of respiratory infections that utilizes platelet-activating factor receptor (PAFR) for firm adherence to host cells. The mucolytic agent S-carboxymethylcysteine (S-CMC) has been shown to exert inhibitory effects against infection by several respiratory pathogens including S. pneumoniae in vitro and in vivo. Moreover, clinical studies have implicated the benefits of S-CMC in preventing exacerbation of chronic obstructive pulmonary disease, which is considered to be related to respiratory infections. In this study, to assess whether the potency of S-CMC is attributable to inhibition of pneumococcal adherence to host cells, an alveolar epithelial cell line stimulated with interleukin-1α was used as a model of inflamed epithelial cells. Despite upregulation of PAFR by inflammatory activation, treatment with S-CMC efficiently inhibited pneumococcal adherence to host epithelial cells. In order to gain insight into the inhibitory mechanism, the effects of S-CMC on PAFR expression were also investigated. Following treatment with S-CMC, PAFR expression was reduced at both mRNA and post-transcriptional levels. Interestingly, S-CMC was also effective in inhibiting pneumococcal adherence to cells transfected with PAFR small interfering RNAs. These results indicate S-CMC as a probable inhibitor targeting numerous epithelial receptors that interact with S. pneumoniae.

Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b.

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

PfbA, a novel plasmin- and fibronectin-binding protein of Streptococcus pneumoniae, contributes to fibronectin-dependent adhesion and antiphagocytosis.

Streptococcus pneumoniae is a major causative agent of mortality throughout the world. The initial event in invasive pneumococcal disease is the attachment of pneumococci to epithelial cells in the upper respiratory tract. Several bacterial proteins can bind to host extracellular matrix proteins and function as adhesins and invasins. To identify adhesins or invasins on the pneumococcal cell surface, we searched for several proteins with an LPXTG anchoring motif in the whole-genome sequence of Streptococcus pneumoniae and identified one, which we called PfbA (plasmin- and fibronectin-binding protein A), that bound to human serum proteins. Immunofluorescence microscopy and fluorescence-activated cell sorter analysis revealed that PfbA was expressed on the pneumococcal cell surface. A DeltapfbA mutant strain was only half as competent as the wild-type strain at adhering to and invading lung and laryngeal epithelial cells. In addition, epithelial cells infected with DeltapfbA showed morphological changes, including cell flattening and a loss of microvilli, that did not occur in cells infected with the wild-type strain. The mutant strain also exhibited a weaker antiphagocytotic activity than wild type in human peripheral blood. Moreover, the growth of wild-type bacteria in human whole blood containing anti-PfbA antibodies was reduced by approximately 50% after 3 h compared with its growth without the antibody. These results suggest that PfbA is an important factor in the development of pneumococcal infections.