Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway

Abstract

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.

O-Acetylation of Peptidoglycan Is Required for Proper Cell Separation and S-layer Anchoring in Bacillus anthracis

“…In the host, B. anthracis remodels its peptidoglycan as suggested by the abundant production of peptidoglycan hydrolases during infection (47). Because purified B. anthracis peptidoglycan stimulates an inflammatory response in vitro (48), controlling the composition of muropeptides shed during multiplication in the host may enable better survival of the pathogen. Indeed, the detection of MurNAc by Nod2, the eukaryotic intracellular muropeptide receptor, is altered by the conversion of MurNAc into anhydro-MurNAc (53).…”

Toxin Inhibition of Antimicrobial Factors Induced by Bacillus anthracis Peptidoglycan in Human Blood

“…B. anthracis EF, LF, and PA were purchased from List Biological Laboratories (Campbell, CA). B. anthracis Sterne BaPGN was isolated as described previously (20,21). Fluorochrome-conjugated antibodies to human proteins and other flow cytometry-related items were purchased from the following vendors: anti-human CD3 (phycoerythrin [PE]-Alexa Fluor 610) and CD19 (allophycocyanin [APC]-Alexa Fluor 750) and Pacific blue-conjugated streptavidin from Invitrogen, Carlsbad, CA; anti-human CD16b (fluorescein isothiocyanate [FITC]) from GeneTex, Irvine, CA; anti-human CD14 (peridinin chlorophyll protein [PerCP]-Cy5.5), IL-1␣ (FITC), IL-1␤ (PE), TNF-␣ (PE-Cy7), IL-6 (Alexa Fluor 700), and brefeldin A from eBioscience, San Diego, CA; biotin-conjugated anti-human CD16b from Exbio, San Diego, CA; anti-human MIP-1␤ (PE-Cy7) from BD Pharmingen, San Diego, CA; anti-human IL-8 (APC) from BioLegend, San Diego, CA; anti-human GRO-␣ (PE), MIP-1␣ (APC), and anthrax toxin receptor 2 (ANTXR2/CMG2) and APC-conjugated streptavidin from R&D Systems, Minneapolis, MN; anti-human TEM8/ANTXR1 from Abcam, Cambridge, MA; and recombinant human GRO-␣ (CXCL1), CXCL10 (IP-10), and MCP1 (CCL2) from PeproTech, Rocky Hill, NJ.…”

“…In the case of B. anthracis, peptidoglycan (BaPGN) is released during growth in blood and has been shown to be a pathogen-associated molecular pattern (PAMP) (20). Further, we have shown that highly purified BaPGN activates the p38 MAPK pathway in monocytes (20,21), and BaPGN polymers are taken up by mononuclear cells, causing monocytes to produce interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-␣), IL-6, and IL-1␤ (20,22). Thus, BaPGN is detected by monocytes and, upon phagocytosis, triggers a response that could assist in clearing the organism from the host.…”

Cellular Signaling Molecules Associated with Peptidoglycan-Induced CCL3 Up-Regulation

“…At nonmucosal sites like vasculature, PGN is recognized by Toll-like receptors (TLRs) expressed by immune cells (e.g. macrophages) (Yoshimura et al, 1999), which triggers intracellular signal transduction pathways, resulting in the production of pro-infl ammatory cytokines (Langer et al, 2008) and chemokines (Wang et al, 2000). Since PGN is presumed to be an additional proinfl ammatory factor in atherosclerotic lesions (Laman et al, 2002), elucidation of the mechanism of action of PGN-induced infl ammatory responses will broaden current knowledge of the roles of bacterial PAMPs in atherogenesis.…”