Bacterial Lipoproteins Research Articles

Induction of apoptosis in human neutrophils by Mycobacterium tuberculosis is dependent on mature bacterial lipoproteins

Modulation of immune cell apoptosis is a key evasion strategy utilized by Mycobacterium tuberculosis (Mtb). To be able to multiply within macrophages, the bacterium delays apoptosis and down-regulates pro-inflammatory activation in these cells, whereas apoptosis is rapidly induced in the potently bactericidal neutrophils. Initial host-pathogen interactions between neutrophils and Mtb, subsequently leading to apoptosis, need to be investigated to understand the early features during Mtb infections. Opsonized Mtb were readily phagocytosed, and the immuno-mediated phagocytosis triggered early activation of anti-apoptotic Akt in the neutrophils but the bacteria still induced apoptosis to the same extent as non-phagocytosed Mtb. Mtb-induced apoptosis was strictly dependent on NADPH oxidase-generated reactive oxygen species, compounds shown to damage lysosomal granules. Despite this, we found no involvement of damaged azurophilic granules in Mtb-induced apoptosis in human neutrophils. Instead, the Mtb-induced apoptosis was p38 MAPK dependent and induced through the mitochondrial pathway. Moreover, Mtb deficient of mature lipoproteins lacked the determinants required for induction of neutrophil apoptosis. These results show that Mtb exert a strong intrinsic capacity to induce apoptosis in neutrophils that is capable of overcoming the anti-apoptotic signaling in the cell.

A TLR2 ligand suppresses inflammation by modulation of chemokine receptors and redirection of leukocyte migration

AbstractToll-like receptors orchestrate rapid local protective innate-immune responses to invading pathogens and optimize leukocyte priming of subsequent adaptive responses. Paradoxically, systemic excess of the TLR2 ligand, bacterial lipoprotein (BLP), suppresses peripheral inflammatory responses. Here, we demonstrate that this phenomenon is regulated via the TLR2-dependent, cell-autonomous down-regulation of inflammatory chemokine receptor expression on a variety of leukocyte subsets. Remarkably, BLP mediated no effect on constitutive chemokine receptor expression. By tracking adoptively transferred wild-type and TLR2−/− leukocytes in vivo, we observed that BLP mediated chemokine receptor switching directed leukocytes away from inflamed sites toward secondary lymphoid organs. These data highlight a novel role for TLR ligands, such as BLP, in regulating leukocyte retention and migration away from innate immune lesions via discrete constitutive and inflammatory chemokine receptor regulation.

Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB

Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detail. However, it remained unclear how Lol factors interact with each other to conduct very efficient lipoprotein transfer in the periplasm where ATP is not available. To address this issue, a photo-reactive phenylalanine analogue, p-benzoyl-phenylalanine, was introduced at various positions of LolA and LolB, of which the overall structures are very similar and comprise an incomplete beta-barrel with a hydrophobic cavity inside. Cells expressing LolA or LolB derivatives containing the above analogue were irradiated with UV for in vivo photo-cross-linking. These analyses revealed a hot area in the same region of LolA and LolB, through which LolA and LolB interact with each other. This area is located at the entrance of the hydrophobic cavity. Moreover, this area in LolA is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between LolA and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of LolA and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of lipoproteins through Lol proteins occurs in a mouth-to-mouth manner.

Galectin-3 deficiency reduces the severity of experimental autoimmune encephalomyelitis (99.5)

Abstract Galectin-3 (Gal-3) is a member of β-galactoside-binding lectin family and plays an important role in inflammatory. However, the precise role of Gal-3 in autoimmune diseases remains obscure. We have investigated the functional role of Gal-3 in experimental autoimmune encephalomyelititis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Gal-3 deficient (Gal-3-/-) mice developed significantly milder EAE and markedly reduced leukocyte infiltration in the CNS compared with similarly treated wild-type (WT) mice. Gal-3-/- mice also contained fewer monocytes and macrophages but more apoptotic cells in the CNS than did WT mice. Following Ag stimulation in vitro, lymph node cells from the immunized Gal-3-/- mice produced less IL-17 and IFN-γ than did those of the WT mice. In contrast, Gal-3-/- mice produced more serum IL-10, IL-5 and IL-13 and contained higher frequency of Foxp3+ regulatory T cells in the CNS than did the WT mice. Furthermore, bone marrow-derived dendritic cells from Gal-3-/- mice produced more IL-10 in response to LPS or bacterial lipoprotein than did WT marrow-derived dendritic cells. Moreover, Gal-3-/- dendritic cells induced AG-specific T cells to produce more IL-10, IL-5 and IL-12, but less IL-17 than did WT dendritic cells. Taken together, our data demonstrate that Gal-3 plays an important disease-exacerbating role in EAE through its multifunctions roles in preventing cell apoptosis and increasing IL-17 and IFN-γ synthesis, but decreasing IL-10 production.

Variability of In Vitro Macrophage Activation by Commercially Diverse Bulk Echinacea Plant Material is Due Predominantly to Bacterial Lipoproteins and Lipopolysaccharides

We previously reported that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea and other botanicals depends on bacterial lipopolysaccharides and Braun type bacterial lipoproteins [1]. We determined the contribution made by these bacterial components to the overall immune enhancing activity detected in E. purpurea and E. angustifolia from bulk root and aerial material obtained from six major growers/suppliers in North America. Substantial variation in activity (up to 200-fold) was observed in extracts of these materials when tested in two monocyte/macrophage cell lines. The majority of activity was negated by treatment with agents that target bacterial lipoproteins (lipoprotein lipase) and lipopolysaccharides (polymyxin B). Experiments comparing the activity of freeze dried, freshly harvested Echinacea plants with those harvested and dried using various commercially relevant conditions, suggest that post-harvesting procedures do not substantially contribute to the variation observed in the commercial material. Acknowledgements: This research was partially funded by grants from the National Institutes of Health RO1 AT002360 (NCAAM) to DSP and the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58-6408-7-012.

Polyanionic Microbicides Modify Toll-Like Receptor-Mediated Cervicovaginal Immune Responses

Topical microbicides are being developed as a preventative approach to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1) and other infections. For them to be efficacious, it is believed that they should avoid inducing inflammation while allowing the vaginal epithelium to initiate protective Toll-like receptor (TLR)-mediated innate responses against pathogens. In this study, human cervical and vaginal epithelial cells were exposed to polyanionic HIV entry inhibitors and the following synthetic TLR ligands: (i) the bacterial lipoprotein Pam(3)CSK(4), binding cell surface TLR1/TLR2; (ii) macrophage activating lipopeptide 2 (MALP-2), binding cell surface TLR2/TLR6; and (iii) the viral double-stranded RNA analog poly(I:C), recognized by intracellular TLR3. Cell activation was assessed by nuclear factor kappaB (NF-kappaB) reporter gene transactivation and cytokine production. In spite of enhancing TLR-triggered NF-kappaB activation, the polyanionic microbicide compounds dextran sulfate and polystyrene sulfonate significantly inhibited TLR-mediated cytokine production. They decreased cytokine mRNA and protein levels of proinflammatory (interleukin-8 [IL-8] and IL-1beta) and antiviral (beta interferon) cytokines following epithelial cell stimulation with Pam(3)CSK(4), MALP-2, or poly(I:C). These activities were associated with the sulfate/sulfonate moieties of the polyanionic compounds, since the unsulfated dextran control did not show any effects. Our data demonstrate that these microbicide compounds are capable of selectively interfering with TLR-mediated epithelial responses at different points in their signaling pathways and underscore the importance of expanding the assessment of microbicide compatibility with vaginal innate immune function. Further studies are warranted to determine the impact of this interference on HIV-1 transmission risk.

Galectin-3 Deficiency Reduces the Severity of Experimental Autoimmune Encephalomyelitis

Galectin-3 (Gal-3) is a member of the beta-galactoside-binding lectin family and plays an important role in inflammation. However, the precise role of Gal-3 in autoimmune diseases remains obscure. We have investigated the functional role of Gal-3 in experimental autoimmune encephalomyelitis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Gal-3 deficient (Gal-3-/-) mice developed significantly milder EAE and markedly reduced leukocyte infiltration in the CNS compared with similarly treated wild-type (WT) mice. Gal-3-/- mice also contained fewer monocytes and macrophages but more apoptotic cells in the CNS than did WT mice. Following Ag stimulation in vitro, lymph node cells from the immunized Gal-3-/- mice produced less IL-17 and IFN-gamma than did those of the WT mice. In contrast, Gal-3-/- mice produced more serum IL-10, IL-5, and IL-13 and contained higher frequency of Foxp3+ regulatory T cells in the CNS than did the WT mice. Furthermore, bone marrow-derived dendritic cells from Gal-3-/- mice produced more IL-10 in response to LPS or bacterial lipoprotein than did WT marrow-derived dendritic cells. Moreover, Gal-3-/- dendritic cells induced Ag-specific T cells to produce more IL-10, IL-5, and IL-12, but less IL-17, than did WT dendritic cells. Taken together, our data demonstrate that Gal-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-gamma synthesis, but decreasing IL-10 production.

RP105 Facilitates Macrophage Activation by Mycobacterium tuberculosis Lipoproteins

RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

2SA2-02 細菌型リポ蛋白質の選別・輸送・膜局在化を司るLolシステムの構造と機能(2SA2 タンパク質の膜透過輸送の最前線,第47回日本生物物理学会年会)

2SA2-02 細菌型リポ蛋白質の選別・輸送・膜局在化を司るLolシステムの構造と機能(2SA2 タンパク質の膜透過輸送の最前線,第47回日本生物物理学会年会)

Toll-like receptor and antimicrobial peptide expression in the bovine endometrium

BackgroundThe endometrium is commonly infected with bacteria leading to severe disease of the uterus in cattle and humans. The endometrial epithelium is the first line of defence for this mucosal surface against bacteria and Toll-like receptors (TLRs) are a critical component of the innate immune system for detection of pathogen associated molecular patterns (PAMPs). Antimicrobial peptides, acute phase proteins and Mucin-1 (MUC-1) also provide non-specific defences against microbes on mucosal surfaces. The present study examined the expression of innate immune defences in the bovine endometrium and tested the hypothesis that endometrial epithelial cells express functional receptors of the TLR family and the non-specific effector molecules for defence against bacteria.MethodsBovine endometrial tissue and purified populations of primary epithelial and stromal cells were examined using RT-PCR for gene expression of TLRs, antimicrobial peptides and MUC-1. Functional responses were tested by evaluating the secretion of prostaglandin E2 and acute phase proteins when cells were treated with bacterial PAMPs such as bacterial lipopolysaccharide (LPS) and lipoproteins.ResultsThe endometrium expressed TLRs 1 to 10, whilst purified populations of epithelial cells expressed TLRs 1 to 7 and 9, and stromal cells expressed TLRs 1 to 4, 6, 7, 9 and 10. The TLRs appear to be functional as epithelial cells secreted prostaglandin E2 in response to bacterial PAMPs. In addition, the epithelial cells expressed antimicrobial peptides, such as Tracheal and Lingual Antimicrobial Peptides (TAP and LAP) and MUC-1, which were upregulated when the cells were treated with LPS. However, the epithelial cells did not express appreciable amounts of the acute phase proteins haptoglobin or serum amyloid A.ConclusionEpithelial cells have an essential role in the orchestration of innate immune defence of the bovine endometrium and are likely to be the key to prevention of endometrial infection with bacteria.

Adaptor Protein Lnk Inhibits C-Fms Mediated Macrophage Function

The macrophage colony-stimulating factor receptor (c-Fms) plays an important role in proliferation, differentiation and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. One such protein is the adaptor Lnk that negatively regulates several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain and a pleckstrin homology domain. The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. In this study, we investigated the ability of Lnk to interact and modulate the function of c-Fms. In order to determine if Lnk can bind to c-Fms, immunoprecipitation was performed with lysates from 293T cells co-transfected with the cDNAs for c-Fms and Lnk. Only after exposure to M-CSF, Lnk bound to c-Fms, and binding was dependent on an intact SH2 domain. To elucidate further if Lnk exhibits biological and functional effects on macrophages, we examined both in-vitro differentiated macrophages derived from the bone marrow and also macrophages harvested from peritoneum from Lnk deleted (KO) and wild type (WT) mice. These cells appeared to be at a similar stage of differentiation because expression levels of myeloid and macrophage surface markers such as F4/80, CD11b and CD11c were the same in both bone marrow-derived and peritoneum-derived macrophages from Lnk KO and WT mice. Clonogenic assays demonstrated that the number of M-CFUs in the bone marrow were elevated in Lnk KO as compared to WT mice. Furthermore, the M-CSF-induced phosphorylation of AKT in these Lnk KO macrophages was increased and prolonged compared to WT macrophages. This was associated with prominent up-regulation of c-Fms in macrophages from Lnk KO mice. We found that Lnk additionally had several functional effects on bone marrow-derived macrophages. Production of reactive oxygen species (ROS) was dramatically increased in a M-CSF-dependent manner in Lnk KO macrophages upon stimulation with zymosan. In addition, knock-out of Lnk led to altered cytokine production of macrophages: Stimulation with zymosan caused increased levels of TNFalpha and IL-6 in the KO cells, while bacterial lipoproteins (Pam3CSK4) decreased levels of TNFalpha in KO compared to WT macrophages. Last, Lnk inhibited M-CSF-induced migration of macrophages in the Boyden chamber as Lnk KO macrophages showed a significantly higher migration capacity than WT macrophages. In summary, we show for the first time that Lnk can bind to c-Fms and can blunt the stimulation of M-CSF. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

Variability in in Vitro Macrophage Activation by Commercially Diverse Bulk Echinacea Plant Material Is Predominantly Due to Bacterial Lipoproteins and Lipopolysaccharides

We previously reported that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea and other botanicals depends upon bacterial lipopolysaccharides and Braun-type bacterial lipoproteins. We determined the contribution made by these bacterial components to the overall immune-enhancing activity detected in E. purpurea and E. angustifolia bulk root and aerial material obtained from six major growers/suppliers in North America. Substantial variation in activity (up to 200-fold) was observed in extracts of these materials when tested in two monocyte/macrophage cell lines. The majority of activity was negated by treatment with agents that target bacterial lipoproteins (lipoprotein lipase) and lipopolysaccharides (polymyxin B). Experiments comparing the activity of freeze-dried, freshly harvested Echinacea plants to those harvested and dried using various commercially relevant conditions suggest that postharvesting procedures do not substantially contribute to the variation observed in the commercial material.

Host Inactivation of Bacterial Lipopolysaccharide Prevents Prolonged Tolerance Following Gram-Negative Bacterial Infection

A transient state of tolerance to microbial molecules accompanies many infectious diseases. Such tolerance is thought to minimize inflammation-induced injury, but it may also alter host defenses. Here we report that recovery from the tolerant state induced by Gram-negative bacteria is greatly delayed in mice that lack acyloxyacyl hydrolase (AOAH), a lipase that partially deacylates the bacterial cell-wall lipopolysaccharide (LPS). Whereas wild-type mice regained normal responsiveness within 14 days after they received an intraperitoneal injection of LPS or Gram-negative bacteria, AOAH-deficient mice had greatly reduced proinflammatory responses to a second LPS injection for at least 3 weeks. In contrast, LPS-primed Aoah- knockout mice maintained an anti-inflammatory response, evident from their plasma levels of interleukin-10 (IL-10). LPS-primed Aoah-knockout mice experiencing prolonged tolerance were highly susceptible to virulent E. coli challenge. Inactivating LPS, an immunostimulatory microbial molecule, is thus important for restoring effective host defenses following Gram-negative bacterial infection in animals.

TLR Expression on Neutrophils at the Pulmonary Site of Infection: TLR1/TLR2-Mediated Up-Regulation of TLR5 Expression in Cystic Fibrosis Lung Disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Methods for the bioinformatic identification of bacterial lipoproteins encoded in the genomes of Gram-positive bacteria

Bacterial lipoproteins are a diverse and functionally important group of proteins that are amenable to bioinformatic analyses because of their unique signal peptide features. Here we have used a dataset of sequences of experimentally verified lipoproteins of Gram-positive bacteria to refine our previously described lipoprotein recognition pattern (G+LPP). Sequenced bacterial genomes can be screened for putative lipoproteins using the G+LPP pattern. The sequences identified can then be validated using online tools for lipoprotein sequence identification. We have used our protein sequence datasets to evaluate six online tools for efficacy of lipoprotein sequence identification. Our analyses demonstrate that LipoP (http://www.cbs.dtu.dk/services/LipoP/) performs best individually but that a consensus approach, incorporating outputs from predictors of general signal peptide properties, is most informative.

β3 Integrin as a Coreceptor for TLR2

Toll-like receptor 2 (TLR2) is one of a family of TLRs involved in recognition of pathogenic molecular motifs. TLR2 signaling is specifically activated by bacterial lipoproteins, such as LTA (lipoteichoic acid) and zymosan, and can be activated by synthetic bacterial lipopeptides (BLPs) consisting of tri- or di-palmitoyl- S -glycerylcysteine and a short peptide, but not mono-acylated peptides. Gerold et al . found that human monocytes (THP-1 cells) produced more inflammatory cytokine in response to the triacylated BLP(SK4) in the presence of serum than in the absence, and, with a version of BLP(SK4) modified with a crosslinker and affinity tags, identified vitronectin as the serum protein responsible for this enhanced response. Priming THP-1 cells with a phorbol ester increased the transcription and abundance of the β3 integrin subunit, which is the receptor for vitronectin, and primed cells plated on vitronectin, but not other extracellular matrix proteins, showed enhanced cytokine production in response to BLP(SK4), as well as to the TLR2 ligands LTA and zymosan, and the TLR4 ligand LPS. Blocking β3 integrin with either function-blocking antibodies or with a snake venom protein inhibited BLP(SK4)-mediated stimulation of cytokine production. When β3 integrin was knocked down with shRNA, the cells produced much less cytokine in response to BLP(SK4); however, the response to LPS, which activates TLR4, was not affected. Cells from patients with Glanzmann thrombasthenia, which have mutations in the ITGA2B gene encoding the integrin α2b or ITGB3 encoding the β3 integrin, lacked β3 on the surface and did not produce cytokines in response to BLP(SK4), suggesting that integrin β3 is essential for TLR2 signaling. β3 was coimmunoprecipitated with TLR2 from THP-1 cells, and BLP(SK4) disrupted this interaction and appeared to destabilize β3, as it became undetectable shortly after stimulation. The authors suggest that vitronectin presents pathogen lipopeptides to a complex of β3 integrin and TLR2, which activates TLR2 and disrupts the complex. G. Gerold, K. A. Ajaj, M. Bienert, H.-J. Laws, A. Zychlinsky, J. L. de Diego, A Toll-like receptor 2-integrin β 3 complex senses bacterial lipopeptides via vitronectin. Nat. Immunol. 9 , 761-768 (2008). [PubMed]

Localization and characterization of prolipoprotein diacylglyceryl transferase (Lgt) critical in bacterial lipoprotein biosynthesis

Lipid modification of proteins is an essential post-translational event that can be targeted for protein engineering and pharmaceutical applications. In this regard, the unique and ubiquitous bacterial N-terminal lipid modification ( N-acyl S-diacylglyceryl modification of N-terminal cysteine) is particularly attractive. It is initiated by phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) and therefore its properties, which remain uninvestigated, largely define the specifics of the modification. A synthetic peptide-substrate (MKATKSAVGSTLAGCSSHHHHHH) with a short hydrophilic h-region, unlike that of the prototypical substrate used so far, demonstrated lack of enzyme's substrate preference based on hydrophobicity, perhaps accounting for a significant number of lipoproteins possessing hydrophilic signal peptides. Solubilization experiments revealed a peripheral and possibly reversible hydrophobic association of Lgt with the inner-membrane on the cytosolic side contradicting its deduced transmembrane topology. Except for heat stability, the soluble enzyme was indistinguishable from the membrane-bound enzyme in kinetic behaviour, indicating that the committed first step of bacterial lipid modification may be aqueous compatible. The direct, more accurate, precise and easier paper electrophoretic assay, designed anew, and Lgt's ready extraction with water or low ionic strength solutions from inverted vesicles could aid better understanding and exploitation of the enzyme and bacterial lipid modification.

Crystal structure of a putative lysostaphin peptidase from Vibrio cholerae.

Peptidoglycan (PGN) constitutes the cell walls of virtually all bacteria, making it a target of the innate immune system. PGN is a polymer of alternating {Beta} (1{yields}4) linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), crossbridged by oligopeptide stems. Lysotaphin type enzymes are believed to cleave the glycl-glycine and glycyl-alanine bonds that occur in glycine-rich cross-bridges. Lysostaphins represent potential anti staphylococcal agents. Specifically, they can eradicate S.aureus nasal colonization in the rat model and are effective in treating methicillin-resistant S. aureus endophthalmitis in rabbits. These enzymes belong to the metalloendopeptidase family and possess a conserved HXH active site motif.

Selective attenuation of Toll-like receptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate

Vascular inflammation is a major atherogenic factor and Toll-like receptor (TLR) 2 ligands, including bacterial and serum lipoproteins, seem to be involved in atherogenesis. On this basis, we analysed the effect of lipoproteins and different lipid components on TLR2-dependent signalling. In TLR2-transfected human embryonic kidney 293 cells and human monocytes, oxidized low-density lipoproteins inhibited nuclear factor (NF)-kappaB-driven transcriptional activity and chemokine gene expression in response to TLR2 ligands. Sphingosine 1-phosphate (S1P) and oxidized palmitoyl-arachidonoyl-phosphatidylcholine, but not lipoprotein-carried lysophospholipids, inhibited TLR2 activation. Silencing experiments in TLR2-transfected 293 cells showed that the S1P-mediated attenuation effect is mediated by S1P receptors type 1 and type 2. To address the physiological significance of these findings, additional experiments were performed in human peripheral blood monocytes and monocyte-derived macrophages. In both cell types, S1P selectively attenuated TLR2 signalling, as NF-kappaB and extracellular signal-regulated kinase activation, but not c-Jun amino terminal kinase phosphorylation, were inhibited by physiologically relevant concentrations of S1P. Moreover, the attenuation of TLR2 signalling was partially reverted by pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and Ras pathways. In addition, S1P inhibited the chemokine gene expression elicited by TLR2, but not by TLR4 ligands. These findings disclose a cross-talk mechanism between lipoprotein components and TLR in which engagement of S1P receptors exert selective attenuation of TLR2-dependent activation via PI3K and Ras signalling. A corollary to these data is that the negative cross-talk of S1P receptors and TLR2 signalling might be involved in the atheroprotective effects of S1P.

Lipoproteins Are Critical TLR2 Activating Toxins in Group B Streptococcal Sepsis

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.