To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

Summary The lipidic envelope of Mycobacterium tuberculosis promotes virulence in many ways, so we developed a lipidomics platform for broad survey of cell walls. Here we report two new databases (MycoMass, MycoMap), 30 lipid fine maps and mass spectrometry datasets that comprise a static lipidome. Further, by rapidly regenerating lipidomic datasets during biological processes, comparative lipidomics provides statistically valid, organism-wide comparisons that broadly assess lipid changes during infection or among clinical strains of mycobacteria. Using stringent data filters, we tracked more than 5,000 molecular features in parallel with few or no false positive molecular discoveries. The low error rates allowed the first chemotaxonomic analyses of mycobacteria, which describe the extent of chemical change in each strain and identified particular strain-specific molecules for use as biomarkers.

Macrophages are the guardians of the innate immune system, recognizing a broad array of pathogen-associated molecular patterns (PAMPs) to initiate immediate defenses and to recruit the adaptive branch of the immune system. Toll-like receptors (TLRs) detect extracellular microbial products, such as lipopolysaccharide, peptidoglycan, lipotechoic acid, and fl agellin (1), whereas surveillance of the cytosol is the task of nucleotide-binding oligomerization domain (NOD) leucine-rich repeat (LRR) proteins. The best-characterized members of the NOD-LRR family are NOD1 and NOD2, which recognize distinct elements of bacterial cell wall peptidoglycan in the cytosol to mount or modulate a proinfl ammatory immune response or to promote apoptosis (2). In mouse macrophages, the NOD-LRR protein Naip5 (Birc1e) restricts intracellular replication of the opportunistic human pathogen Legionella pneumophila (3-5). Naip5 is comprised of three modules: NH 2-terminal baculoviral inhibitor of apoptosis repeats, a central NOD domain, and COOH-terminal LRRs (2). By analogy to other NOD-LRR proteins, the LRR region is thought to recognize microbial products, triggering oligomerization via the NOD domain and activation of a cellular response that is governed by various NH 2terminal eff ector-binding domains (2). Whereas virtually all mice are resistant to L. pneumophila, the A/J strain encodes a naip5 allele that confers susceptibility to infection (3). Whether the

SUMMARY Mycobacterium leprae causes leprosy, and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interactions of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia, and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy.