Internalization of Polymeric Bacterial Peptidoglycan Occurs through Either Actin or Dynamin Dependent Pathways.

Peptidoglycan (PGN), a polymeric glycan macromolecule, is a major constituent of the bacterial cell wall and a conserved pathogen-associated molecular pattern (PAMP) that triggers immune responses through cytosolic sensors. Immune cells encounter both PGN polymers and hydrolyzed muropeptides during infections, and primary human innate immune cells respond better to polymeric PGN than the minimal bioactive subunit muramyl dipeptide (MDP). While MDP is internalized through macropinocytosis and/or clathrin-mediated endocytosis, the internalization of particulate polymeric PGN is unresolved. We show here that PGN macromolecules isolated from Bacillus anthracis display a broad range of sizes, making them amenable for multiple internalization pathways. Pharmacologic inhibition indicates that PGN primarily, but not exclusively, is internalized by actin-dependent endocytosis. An alternate clathrin-independent but dynamin dependent pathway supports 20-30% of PGN uptake. In primary monocytes, this alternate pathway does not require activities of RhoA, Cdc42 or Arf6 small GTPases. Selective inhibition of PGN uptake shows that phagolysosomal trafficking, processing and downstream immune responses are drastically affected by actin depolymerization, while dynamin inhibition has a smaller effect. Overall, we show that polymeric PGN internalization occurs through two endocytic pathways with distinct potentials to trigger immune responses.

C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils.

Neutrophils are the most abundant innate cell population and a key immune player against invading pathogens. Neutrophils can kill both bacterium and spores of Bacillus anthracis, the causative anthrax pathogen. Unlike interactions with professional phagocytes, the molecular recognition of anthrax by neutrophils is largely unknown. In this study, we investigated the role of complement C3 deposition on anthrax particles for neutrophil recognition of bacterium and/or its cell wall peptidoglycan, an abundant pathogen-associated molecular pattern that supports anthrax sepsis. C3 opsonization and recognition by complement receptors accounted for 70-80% of the affinity interactions between neutrophils and anthrax particles at subphysiologic temperatures. In contrast, C3 supported up to 50% of the anthrax particle ingestion under thermophysiologic conditions. Opsonin-dependent low affinity interactions and, to a lower extent, opsonin-independent mechanisms, provide alternative entry routes. Similarly, C3 supported 58% of peptidoglycan-induced degranulation and, to a lower extent, 23% of bacterium-induced degranulation. Interestingly, an opsonin independent mechanism mediated by complement C5, likely through C5a anaphylatoxin, primes azurophilic granules in response to anthrax particles. Overall, we show that C3 deposition supports anthrax recognition by neutrophils but is dispensable for pathogen ingestion and neutrophil degranulation, highlighting immune recognition redundancies that minimize the risk of pathogen evasion.