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Peptidoglycan

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Peptidoglycan is a structural component of the cell wall in both Gram-positive and Gram-negative bacteria. It is a complex polymer consisting of N-acetylglucosamine and N-acetylmuramic acid residues crosslinked by short peptide chains. Peptidoglycan provides mechanical strength and shapes the bacterial cell.

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Lab products found in correlation

Poly 1 c, by InvivoGen (4 mentions) Cpg dna, by InvivoGen (2 mentions) Cyclophosphamide, by Merck Group (2 mentions) Malp 2, by Enzo Life Sciences (2 mentions) Flagellin, by Enzo Life Sciences (2 mentions) Recombinant human and mouse marco, by R&D Systems (2 mentions) Human olr1, by R&D Systems (2 mentions) Imiquimod, by InvivoGen (2 mentions) Interleukin 4 (il 4), by R&D Systems (2 mentions) Il 13, by R&D Systems (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) N acetyl l cysteine nac, by Merck Group (1 mentions) Ammonium pyrrolidinedithiocarbamate pdtc, by Merck Group (1 mentions) Cardiolipin, by Merck Group (1 mentions) Calcein, by Dojindo Laboratories (1 mentions) Lipoteichoic acid, by Merck Group (1 mentions) Peptidoglycan, by Merck Group (1 mentions) Triton x 100, by Thermo Fisher Scientific (1 mentions) L α phosphatidyl dl glycerol, by Merck Group (1 mentions) Bovine serum albumin fraction 5, by Merck Group (1 mentions)

7 protocols using peptidoglycan

1

Immunomodulatory Agents Procurement

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Poly(I:C), Imiquimod, Peptidoglycan and CpG DNA were purchased from Invivogen, San Diego, CA. Malp-2 and Flagellin were purchased from Enzo Life Sciences, Farmingdale, NY. Bovine serum albumin (BSA), Lipopolysaccharide, Poly(I), Poly(C), Dextran Sulfate, and cyclophosphamide were purchased from Sigma-Aldrich, St. Louis, MO. Chloroquine phosphate was purchased from Spectrum, Gardena, CA. Recombinant human and mouse MARCO, and human OLR1, IL-4, and IL-13 were purchased from R&D Systems, Minneapolis, MN.
MacLeod D.T., Nakatsuji T., Wang Z., di Nardo A, & Gallo R.L. (2014). Vaccinia virus binds to the scavenger receptor MARCO on the surface of keratinocytes. The Journal of investigative dermatology, 135(1), 142-150.
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2

Inactivated GAS Immune Response Study

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Heat-killed GAS were prepared by placing bacteria at 65–75°C for 5–10 min; absence of CFU growth was confirmed by plating aliquots on THA. The multiplicity of infection (MOI) of GAS-to-cultured cells (immune and non-immune cells) was 1:1 unless specified. Peritoneal exudate cells (PEC, ~30% macrophages and ~40% B cells), F244 fibrosarcoma, and HEK293 cells were cultured in RPMI containing 10% FBS with penicillin and streptomycin and switched to 2% FBS without antibiotics the day before the experiment. PECs were collected by lavage using 2.5mL PBS or FACS buffer per peritoneum. Cell culture plates were spun at 1500 rpm for 5 min to facilitate contact between GAS and cultured cells. Cells were pre-treated for 1 h with either N-Acetyl-L-cysteine (NAC) or ammonium pyrrolidine dithiocarbamate (PDTC) at a concentration of 0.6 μM and 0.4 μM respectively (Sigma). Final concentration of peptidoglycan was prepared as 1μg/mL (Invivogen).
Washington A J.r., Varki N., Valderrama J.A., Nizet V, & Bui J.D. (2020). Evaluation of interleukin-17D in host immunity to group A Streptococcus infection. Journal of immunology (Baltimore, Md. : 1950), 205(11), 3122-3129.
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3

Antimicrobial Peptide Characterization

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Dimethyl sulfoxide, bovine serum albumin (fraction V), cardiolipin, L-α-phosphatidyl-DL-glycerol, peptidoglycan purified from S. aureus, lysozyme, and lipoteichoic acid purified from S. aureus were purchased from Sigma-Aldrich. Cholesterol, 2-dioleoyl-sn-glycero-3-phosphocholine, and 2-dioleoyl-sn-glycero-3-phosphoethanolamine were purchased from Avanti Polar Lipids Inc. peptidoglycan purified from E. coli was purchased from InvivoGen. Calcein was purchased from Dojindo. Triton X-100 was purchased from Thermo Fisher Scientific. Lipopolysaccharide purified from E. coli 0111:B4 was purchased from List Biological Laboratories, Inc. Synthetic lipid A was purchased from Peptide Institute Inc. Ruby protein gel stain and Any kDTM precast polyacrylamide gels were purchased from Bio-Rad.
Antimicrobial peptides and biotin-labeled antimicrobial peptides were commercially synthesized by Hayashi Kasei, Thermo Fisher Scientific, and the Toray Research Center. C-terminals of the synthetic peptides were modified by amidation. All peptides were initially suspended in Dimethyl sulfoxide.
Manabe T, & Kawasaki K. (2017). D-form KLKLLLLLKLK-NH2 peptide exerts higher antimicrobial properties than its L-form counterpart via an association with bacterial cell wall components. Scientific Reports, 7, 43384.
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4

Neutrophil Stimulation and Gene Expression

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Human neutrophils were isolated and stimulated with medium or
109 HKSA/ml (Heat killed S. aureus or 10
μg/ml peptidoglycan (InvivoGen) for 4 h. Probes for
CRLF2 (Hs00845692_m1) and RPL7(Hs02596927_g1) were from Life technologies.
West E.E., Spolski R., Kazemian M., Yu Z.X., Kemper C, & Leonard W.J. (2016). A TSLP-complement axis mediates neutrophil killing of methicillin-resistant Staphylococcus aureus. Science immunology, 1(5), eaaf8471.
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5

Immunomodulatory Agents Procurement

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Poly(I:C), Imiquimod, Peptidoglycan and CpG DNA were purchased from Invivogen, San Diego, CA. Malp-2 and Flagellin were purchased from Enzo Life Sciences, Farmingdale, NY. Bovine serum albumin (BSA), Lipopolysaccharide, Poly(I), Poly(C), Dextran Sulfate, and cyclophosphamide were purchased from Sigma-Aldrich, St. Louis, MO. Chloroquine phosphate was purchased from Spectrum, Gardena, CA. Recombinant human and mouse MARCO, and human OLR1, IL-4, and IL-13 were purchased from R&D Systems, Minneapolis, MN.
MacLeod D.T., Nakatsuji T., Wang Z., di Nardo A, & Gallo R.L. (2014). Vaccinia virus binds to the scavenger receptor MARCO on the surface of keratinocytes. The Journal of investigative dermatology, 135(1), 142-150.
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6

Antigen-Specific CD4+ T Cell Responses

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The mice were immunized in both feet with either 100 μg/mouse Ovalbumin (OVA, Sigma) or 100 μg/mouse 2W1S peptide (EAWGALANWAVDSA, Genscript) plus 10 μg/mouse LPS (Sigma) emulsified in incomplete Freund’s adjuvant (IFA) as carrier (Sigma). For some experiments, LPS was replaced with either 20 μg/mouse CpG DNA 1826 (Keck Biotechnology Resource Laboratory, Yale Medical School), 50 μg/mouse polyI:C (Invivogen), 20 μg/mouse Peptidoglycan (Invivogen), or 150 μg/mouse heat-inactivated M. tuberculosis (Sigma). The effects of contaminating endotoxin in the OVA preparations were controlled by using endotoxin-free OVA (Hyglos/Biovendor) in key experiments. CD4+ memory responses were induced similarly with the exception that one foot was used for the primary immunization, while the other foot was used for the secondary immunization.
Schenten D., Nish S.A., Yu S., Yan X., Lee H.K., Brodsky I., Pasman L., Yordy B., Wunderlich T., Brüning J.C., Zhao H, & Medzhitov R. (2014). MyD88 signaling in CD4+ T cells is required to overcome suppression by regulatory T cells. Immunity, 40(1), 78-90.
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7

Investigating HIV Infection and Immune Response in Memory CD4 T Cells

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Most experiments involved culture of uninfected or HIV-infected memory CD4 T cells in IL2 (20ng/ml) medium for up to 1 week, followed by data collection. Cells were cultured in 48-well plates (5x105 cells in 1ml medium) with appropriate reagents. The HIV antiviral inhibitor, Saquinavir, was acquired from NIHAIDS Reagent Program and used at 1–10μg/ml. The GrzB/caspase inhibitor, Z-IETD-FMK (Enzo Life Sciences, Farmingdale, NY USA), was used at 10μM. Experiments involving TLR stimulation of memory CD4 T cells utilized synthetic TLR ligands LPS (Sigma, St. Louis, MO USA), Pam3CSK4 (EMD Millipore, Billerica, MA USA), peptidoglycan, Poly(I:C), and Poly(U) (InvivoGen, San Diego, CA USA), and were used at 2–25μg/ml +/− CD3 activation (clone UCHT1 coated at 0.1μg/ml).
Couturier J., Hutchison A.T., Medina M.A., Gingaras C., Urvil P., Yu X., Nguyen C., Mahale P., Lin L., Kozinetz C.A., Schmitz J.E., Kimata J.T., Savidge T.C, & Lewis D.E. (2014). HIV replication in conjunction with granzyme B production by CCR5+ memory CD4 T cells: implications for bystander cell and tissue pathologies. Virology, 0, 175-188.
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