Purified Cell Walls Research Articles

CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan.

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

Interrogation of the contribution of (endo)lysin domains to tune their bacteriolytic efficiency provides a novel clue to design superior antibacterials

Bacteriophage-derived endolysins and bacterial autolysins (hereinafter lysins) represent a completely new class of efficient antibacterials. They prevent the development of bacterial resistance and help protect commensal microbiota, producing cell wall lysis. Here we have investigated whether the acquisition of enzymatic active domains (EADs) and cell wall binding domains (CWBDs) of balancing efficiencies could be a way of tuning natural lysin activity. The concept was applied to produce a chimeric lysin of superior antibacterial capacity using the endolysin Skl and the major pneumococcal autolysin LytA. Combination of the Skl EAD and the cell wall choline-binding domain (CBD) of LytA in the chimera QSLA increased the bacterial killing by 2 logs or more compared to parental enzymes at an equal concentration and extended the substrate range to resistant and emergent pneumococci and other pathogens of the mitis group. Contrarily, QLAS, containing LytA EAD and Skl CBD, was inactive against all tested strains, although domain structures were preserved and hydrolysis of purified cell walls maintained in both chimeras. As a whole, our study provides a novel clue to design superior lysins to fight multidrug-resistant pathogens based on domain selection, and a powerful in-vivo active lysin (QSLA) with promising therapeutic perspectives.

Quantitative enzymatic-mass spectrometric analysis of the chitinous polymers in fungal cell walls

Chitin is an essential structural component of complex and dynamic fungal cell walls. It may be converted by partial or full deacetylation to yield chitosan. Here, we describe a method to quantify N-acetyl d-glucosamine (GlcNAc, A) and d-glucosamine (GlcN, D) units and, thus, total amount and average fraction of acetylation (x̅ FA) of the chitinous polymers by complete enzyme hydrolysis of the polymers followed by mass spectrometric analyses of the monomers. First, the native polymers were isotopically N-acetylated, then enzymatically hydrolyzed to A and R (2H3N-acetyl-d-glucosamine – former D) monomers. Relative abundances of A and R units were used to calculate x̅ FA, and a double-isotopically labeled internal standard R* ([13C2,2H3] N-acetyl-d-glucosamine) monomer was used to calculate the absolute amounts of GlcNAc and GlcN units present in the fungal samples. The method was validated using known chitosan polymers and is suitable for both purified cell walls and whole mycelia.

Extraction and Isolation of Cellulose from Cladodes of Cactus Opuntia Ficus-Indica

Cellulose is the most abundant material in the earth, it is considered a renewable polymer of the cell walls of wood and plant cells, their interest and their use in different field is the purpose of extracting it namely: cellulose native (cotton), the transformation of cellulose into sheets (paper and filter paper), cupric cellulose, zinc cellulose (industrial applications), the functionalization of cellulosic materials in the field of printed electronics, textile, treatment, medicine, and agribusiness. The objective of this study, is the extraction of cellulose at several stages and recovery of a waste based on old cactus rackets, type Opuntia ficus -indica smooth. The cellulose extracted showed good isolation, dispersion and purification of nanofibrils by the removal of oils, mucilage, pectins, hemicellulose and coloring substances. The substitution of ethanol by acetone in the extraction protocol used [1], gave suspensions of purified cell walls is well individualized, on the other hand the cellulose extracted from the beet pulp, chuck pulp prickly pear fruit peel requires a mechanical homogenizer.

Elimination of Deoxynivalenol, Aflatoxin B1, and Zearalenone by Gram-Positive Microbes (Firmicutes).

Mycotoxin contaminations in the feed and food chain are common. Either directly or indirectly, mycotoxins enter the human body through the consumption of food of plant and animal origin. Bacteria with a high mycotoxin elimination capability can reduce mycotoxin contamination in feed and food. Four Gram-positive endospore-forming bacteria (Bacillus thuringiensis AMK10/1, Lysinibacillus boronitolerans AMK9/1, Lysinibacillus fusiformis AMK10/2, and Rummeliibacillus suwonensis AMK9/2) were isolated from fermented forages and tested for their deoxynivalenol (DON), aflatoxin B1 (AFB1), and zearalenone (ZEA) elimination potentials. Notably, the contribution of bacterial cell wall fractions to the observed outstanding ZEA elimination rates was demonstrated; however, the ZEA elimination differed considerably within the tested group of Gram-positive bacteria. It is worth noting that the purified cell wall of L. boronitolerans AMK9/1, L. fusiformis AMK10/2 and B. thuringiensis AMK10/1 were highly efficient in eliminating ZEA and the teichoic acid fractions of B. thuringiensis AMK10/1, and L. fusiformis AMK10/2 could also be successfully used in ZEA binding. The ZEA elimination capacity of viable R. suwonensis AMK9/2 cells was outstanding (40%). Meanwhile, R. suwonensis AMK9/2 and L. boronitolerans AMK9/1 cells produced significant esterase activities, and ZEA elimination of the cell wall fractions of that species did not correlate with esterase activity. DON and AFB1 binding capabilities of the tested bacterial cells and their cell wall fractions were low, except for B. thuringiensis AMK10/1, where the observed high 64% AFB1 elimination could be linked to the surface layer (S-layer) fraction of the cell wall.

Peptidoglycan compositional analysis of Mycobacterium smegmatis using high-resolution LC–MS

Peptidoglycan (PG) is the exoskeleton of bacterial cells and is required for their viability, growth, and cell division. Unlike most bacteria, mycobacteria possess an atypical PG characterized by a high degree of unique linkages and chemical modifications which most likely serve as important determinants of virulence and pathogenesis in mycobacterial diseases. Despite this important role, the chemical composition and molecular architecture of mycobacterial PG have yet to be fully determined. Here we determined the chemical composition of PG from Mycobacterium smegmatis using high-resolution liquid chromatography-mass spectrometry. Purified cell walls from the stationary phase were digested with mutanolysin and compositional analysis was performed on 130 muropeptide ions that were identified using an in silico PG library. The relative abundance for each muropeptide ion was measured by integrating the extracted-ion chromatogram. The percentage of crosslink per PG subunit was measured at 45%. While both 3→3 and 4→3 transpeptide cross-linkages were found in PG dimers, a high abundance of 3→3 linkages was found associated with the trimers. Approximately 43% of disaccharides in the PG of M. smegmatis showed modifications by acetylation or deacetylation. A significant number of PG trimers are found with a loss of 41.00 amu that is consistent with N-deacetylation, whereas the dimers show a gain of 42.01 amu corresponding to O-acetylation of the PG disaccharides. This suggests a possible role of PG acetylation in the regulation of cell wall homeostasis in M. smegmatis. Collectively, these data report important novel insights into the ultrastructure of mycobacterial PG.

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time

Methods for bacterial detection are needed to advance the infection research and diagnostics. Based on conformation-sensitive fluorescent tracer molecules, optotracing was recently established for dynamic detection and visualization of structural amyloids and polysaccharides in the biofilm matrix of gram-negative bacteria. Here, we extend the use of optotracing for detection of gram-positive bacteria, focussing on the clinically relevant opportunistic human pathogen Staphylococcus aureus. We identify a donor-acceptor-donor-type optotracer, whose binding-induced fluorescence enables real-time detection, quantification, and visualization of S. aureus in monoculture and when mixed with gram-negative Salmonella Enteritidis. An algorithm-based automated high-throughput screen of 1920 S. aureus transposon mutants recognized the cell envelope as the binding target, which was corroborated by super-resolution microscopy of bacterial cells and spectroscopic analysis of purified cell wall components. The binding event was essentially governed by hydrophobic interactions, which permitted custom-designed tuning of the binding selectivity towards S. aureus versus Enterococcus faecalis by appropriate selection of buffer conditions. Collectively this work demonstrates optotracing as an enabling technology relevant for any field of basic and applied research, where visualization and detection of S. aureus is needed.

Cell Wall Integrity of Pulse Modulates the in Vitro Fecal Fermentation Rate and Microbiota Composition.

The physical structure of type 1 resistant starch (RS 1) could influence the metabolite production and stimulate the growth of specific bacteria in the human colon. In the present study, we isolated intact cotyledon cells from pinto bean seeds as whole pulse food and RS 1 model and obtained a series of cell wall integrities through controlled enzymolysis. In vitro human fecal fermentation performance and microbiota responses were tested, and we reported that the cell wall integrity controls the in vitro fecal fermentation rate of heat-treated pinto bean cells. The concentration of butyrate produced by pinto bean cell fermentation enhanced with weakened cell wall integrity, and certain beneficial bacterial groups such as Blautia and Roseburia genera were remarkably promoted by pinto bean cells with damaged cell wall integrity. However, the intact cell sample had a shape more similar to microbiota composition with the purified cell wall polysaccharides, rather than the damaged cells.

Surfactant-Induced Competitive Displacement of Potato Pectin-Protein Conjugate from the Air-Water Interface.

Potato pectin contains some proteinaceous components and exhibits emulsifying and emulsion stabilizing abilities. The objective of this study was to elucidate the effect of the pectin moiety of the pectin-protein conjugate present in pectic extracts from potato tubers on their interfacial properties. Potato pectin was extracted from highly purified cell wall materials to avoid the contamination of unconjugated proteins. The abilities of the potato pectin to adsorb to graphite surfaces and to resist surfactant-induced competitive displacement from the air-water interface were investigated using atomic force microscopy. The pectin moiety of the potato pectin was capable of adsorbing to graphite surfaces even after alkali treatment. Furthermore, the potato pectin exhibited enhanced resistance to surfactant-induced competitive displacement from the interface as a result of the formation of network structures of self-assembled pectin moieties at the interface. The present results suggest the importance of the pectin moiety with regard to the interfacial properties of potato pectin.

Synergy Between Two Chimeric Lysins to Kill Streptococcus pneumoniae.

Phage lysins constitute a new generation of antimicrobials that are becoming a promising alternative and complementation to current antibiotic therapies, which are nowadays called into question by the increasing numbers of multiresistant bacteria. Streptococcus pneumoniae is a leading human pathogen causing serious infectious diseases in children and adults. Within the host-parasite interplay system of pneumococcus and its phages, several antipneumococcal lysins have been described and, among them, chimeric lysins Cpl-711 and PL3 stand out for their potent bactericidal activities. Here, evidence is presented on the synergistic cooperation of the catalytically diverse lysins Cpl-711 and PL3 in different assays, like purified cell wall enzymatic degradation, in vitro bacterial cell growth inhibition, and killing of both planktonic and biofilm grown cells. Synergy between Cpl-711 and PL3 has been shown to reduce the amount of enzyme necessary to inhibit growth in checkerboard assays with a sum of fractional inhibitory concentrations ≤0.5 for all pneumococcal strains tested, while also significatively increasing bactericidal effect by ≥2 logs with respect to the sum of activities of Cpl-711 and PL3 individual treatments. Moreover, the combination of these two lysins showed synergy in an adult zebrafish model of pneumococcal infection. This study consolidates the possibility of formulating highly efficient and synergistic antibacterial enzymes that could improve our ability to fight multiresistant bacterial infections.

PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae.

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.

Pear ripeness and tissue type impact procyanidin-cell wall interactions

Procyanidins and cell walls were extracted from pear at ripe and overripe stages in order to investigate the impact of ripening stage on their association. Procyanidin composition and structure remained stable at the overripe stage. Mid Infrared Spectroscopy (MIR) discriminated cell wall-procyanidin complex from initial purified cell wall material (CWM). Interactions between procyanidins and CWM isolated from the whole flesh (FL), parenchyma cells (PC), stone cells (ST) and skin (SK) at ripe and overripe stage were characterized using UV–Vis spectrometry using Langmuir isotherm formulation and Isothermal Titration Microcalorimetry (ITC). The affinity between procyanidins and CWM decreased as follows: PC > FL > ST > SK. The proportion of bound procyanidins increased at the overripe stage for all CWM and the maximal saturation level was obtained for overripe FL and ST. ITC indicated that associations between pear cell walls and procyanidins involved hydrogen bonds and mainly hydrophobic interactions for overripe PC.

Exposure of Staphylococcus aureus to Targocil Blocks Translocation of the Major Autolysin Atl across the Membrane, Resulting in a Significant Decrease in Autolysis.

Peptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptible Staphylococcus aureus strain led to a rapid shut down of cellular autolysis. Targocil II, which targets the TarH subunit of TarGH, also resulted in a drastic decrease in autolysis. Here, we address the mechanism of targocil-mediated decreased autolysis. The mechanism is WTA dependent since targocil treatment decreased autolysis in methicillin-resistant strains but not in a WTA-deficient mutant. Similar to cellular autolysis, autolysin-retaining crude cell walls isolated from targocil-treated cells had vastly decreased autolytic activity compared to those from untreated cells. Purified cell walls from control and targocil-treated cells, which lack autolytic activity, were similarly susceptible to lysozyme and lysostaphin and had similar O-acetyl contents, indicating that targocil treatment did not grossly alter PG structure and chemistry. Purified cell walls from targocil-treated cells were highly susceptible to autolysin extracts, supporting the notion that targocil treatment led to decreased autolysin in the crude cell walls. Quantitative real-time PCR analysis revealed that the decrease in autolysis in the targocil-exposed cells was not due to transcriptional repression of the autolysin genes atl, lytM, lytN, and sle1 Zymographic analysis of peptidoglycan hydrolase profiles showed a deficiency of cell surface autolysins in targocil-treated cells but higher activity in cell membrane fractions. Here, we propose that the untranslocated WTA molecules in the targocil-exposed cells sequester Atl at the membrane, resulting in significantly decreased autolysis.

Preparation of Sedimented Wine Yeast Derived Products for Potential Application in Food and Feed Industry

The aim of this study was to obtain preparations of sedimented wine yeast, which is an unutilised waste from the production of fruit wines in Poland. Yeast extracts, cell wall preparations and purified cell wall preparations were obtained as a result of complete yeast cells fractionation using initial phase of spontaneous autolysis and performed with various process promoters, such as NaCl, glucose, ethanol, acetic acid and lactic acid at different concentrations and in different combinations. The influence of applied autolysis promotors on protein recovery from yeast extracts and protein content in cell wall preparations was assessed. As an important property of yeasts cell wall preparation the influence of alkaline isolation and purification with acetic on the β-glucan content in cell wall were determined. The ability of yeast preparations to bind fat and water was also assessed. Sedimented wine yeast was characterised by low sensitivity to the autolysis process, as from 100 g of dry yeast, depending on the autolysis promoter used, 6.6–10.5 g of protein was recovered. The highest β-glucan content—35.0% in dry matter was assayed in purified cell wall preparation of sedimented wine yeast obtained after autolysis with 0.5% NaCl added to the autolysing mixture. The preparation’s water-binding and fat-binding capacities amounted to 304.8 and 82.9% respectively. Further research in applications obtained yeast products from new raw material (sedimented wine yeast) in food and feed industries must be investigated.

Fission Yeast Cell Wall Analysis.

The Schizosaccharomyces pombe cell wall is a rigid exoskeletal structure mainly composed of interlinked glucose polysaccharides and galactomannoproteins. It is essential for survival of the fission yeast, as it prevents cells from bursting from internal turgor pressure and protects them from mechanical injuries. Additionally, the cell wall determines the cell shape and, therefore, a better knowledge of cell wall structure and composition could provide valuable data in S. pombe morphogenetic studies. Here, we provide information about this structure and the current reliable methods for rapid analysis of the cell wall polymers by specific enzymatic and chemical degradations of purified cell walls.

Isolation of Plasmodesmata.

Plasmodesmata (PD) are plasma membrane lined pores that cross the plant cell wall and connect adjacent cells. Plasmodesmata are composed of elements of the endoplasmic reticulum, plasma membrane, cytosol, and cell wall and thus, as multicomposite structures that are embedded in the cell wall, they are notoriously difficult to isolate from whole plant tissue. However, understanding PD structure, function, and regulation necessitates identification of their molecular components and therefore proteomic and lipidomic analyses of PD fractions are an essential strategy for plasmodesmal biology. Here we outline a simple two-step purification procedure that allows isolation of PD-derived membranes from Arabidopsis suspension cells. The method involves isolation of purified cell wall fragments containing intact PD which is followed by enzymatic degradation of the cell wall to release the PD. This membrane-rich fraction can be subjected to protein and lipid extraction for molecular characterization of PD components. The first step of this procedure involves the isolation of cell wall fragments containing intact PD, free from contamination from other cellular compartments. Purified PD membranes are then released from the cell wall matrix by enzymatic degradation. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates HUVEC proteins related to inflammatory and stress responses

Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this “semi-professional” immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection. SignificanceInvasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial response are unknown. The present work employs a discovery-driven proteomics approach to reveal the role of one important cell wall polysaccharide of A. fumigatus, galactosaminogalactan, in the HUVEC interaction and the consequent mechanisms of endothelial activation. This is the first report of the overall panel of proteins related to the HUVEC response to a specific and purified cell wall component of the angioinvasive fungus A. fumigatus.

Decreased Cell Wall Galactosaminogalactan in Aspergillus nidulans Mediates Dysregulated Inflammation in the Chronic Granulomatous Disease Host.

Invasive aspergillosis is a major threat to patients suffering from impaired neutrophil function, with Aspergillus fumigatus being the most common species causing this life-threatening condition. Patients with chronic granulomatous disease (CGD) not only develop infections with A. fumigatus, but also exhibit a unique susceptibility to infection with the normally nonpathogenic species Aspergillus nidulans. In this study, we compared the inflammatory cytokine response of peripheral blood mononuclear cells (PBMCs) from healthy and CGD patients to these two fungal species. CGD patients displayed evidence for a chronic hyperinflammatory state as indicated by elevated plasma IL-1β and TNF-α levels. PBMCs isolated from CGD patients secreted higher levels of IL-1β and TNF-α in response to A. nidulans as compared with A. fumigatus. The presence or absence of melanin in the cell wall of A. nidulans did not alter the cytokine release by healthy or CGD PBMCs. In contrast, A. fumigatus mutants lacking melanin stimulated higher levels of proinflammatory cytokine release from healthy, but not CGD PBMCs. Purified cell wall polysaccharides of A. nidulans induced a much higher level of IL-1β secretion by CGD PBMCs than did cell wall polysaccharides isolated from A. fumigatus. Using modified A. nidulans strains overexpressing galactosaminogalactan, we were able to show that the increased secretion of inflammatory cytokines by CGD PBMCs in response to A. nidulans are a consequence of low levels of cell wall-associated galactosaminogalactan in this species.

Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential of that reported for C. albicans. In addition, we propose that purified cell wall mannans can be used as antagonist to block specific receptors on innate immune cells.

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues.

Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. This protocol describes how to prepare purified cell wall from Streptococcus pneumoniae, detect its distribution in animal tissues, and study the tissue response using the placenta and fetal brain as examples.