SummaryReactive nitrogen species (RNS) play an essential role in host defence against Mycobacterium tuberculosis (MTB) in the mouse model of tuberculosis (TB), as evidenced by the increased susceptibility of mice deficient in the inducible isoform of nitric oxide synthase (NOS2). In contrast, the role of reactive oxygen species (ROS) in protection against MTB is less clear, and mice defective in the ROS-generating phagocyte NADPH oxidase (Phox) are relatively resistant. This suggests that MTB might possess efficient mechanisms to evade or counter the phagocyte oxidative burst, effectively masking the impact of this host defence mechanism. In order to assess the role of ROS detoxification pathways in MTB virulence, we generated a katG null mutant of MTB, deficient in the KatG catalase-peroxidase-peroxynitritase, and evaluated the mutant's ability to replicate and persist in macrophages and mice. Although markedly attenuated in wild-type C57Bl/6 mice and NOS2 -/-mice, the D D D D katG MTB strain was indistinguishable from wildtype MTB in its ability to replicate and persist in gp91 Phox-/-mice lacking the gp91 subunit of NADPH oxidase. Similar observations were made with murine bone marrow macrophages infected ex vivo : growth of the D D D D katG MTB strain was impaired in macrophages from C57Bl/6 and NOS2 -/-mice, but indistinguishable from wild-type MTB in gp91 Phox-/-macrophages. These results indicate that the major role of KatG in MTB pathogenesis is to catabolize the peroxides generated by the phagocyte NADPH oxidase; in the absence of this host antimicrobial mechanism, KatG is apparently dispensable.

Exposure of bone marrow–derived macrophages (BMDMs) to low concentrations of Bacillus anthracis lethal toxin (LT), whose catalytic subunit is lethal factor (LF), results in induction of a robust apoptotic response dependent on activation of Toll-like receptor (TLR)4. A similar TLR4-dependent apoptotic response is observed when BMDMs are infected with live B. anthracis (Sterne strain). However, TLR4 is considered to be a specific signaling receptor for lipopolysaccharide (LPS), a typical product of gram-negative bacteria, whereas B. anthracis is gram-positive. To understand how B. anthracis can activate TLR4, we analyzed its culture supernatants and found them to contain a potent TLR4-stimulating activity that can also induce apoptosis in macrophages in which the antiapoptotic p38 MAP kinase (whose activation is prevented by LF) was inhibited. Purification of this activity suggested it consists of anthrolysin O (ALO), a member of the cholesterol-dependent cytolysin (CDC) family. We show that recombinant ALO can activate TLR4 in a manner independent of LPS contamination and, together with LT, can induce macrophage apoptosis. We also provide genetic evidence that ALO is required for induction of macrophage apoptosis in response to infection with live B. anthracis and that other CDC family members share the ability to activate TLR4.

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.

Nerve damage is the hallmark of Mycobacterium leprae infection, which results from M. leprae invasion of the Schwann cell of the peripheral nervous system. We have recently shown that the laminin-2 isoform, specially the G domain of laminin ␣2 chain, on the Schwann cell-axon unit serves as an initial neural target for M. leprae. However, M. leprae surface molecules that mediate bacterial invasion of peripheral nerves are entirely unknown. By using human ␣2 laminins as a probe, a major 28-kDa protein in the M. leprae cell wall fraction that binds ␣2 laminins was identified. After N-terminal amino acid sequence analysis, PCR-based strategy was used to clone the gene that encodes this protein. Deduced amino acid sequence of this M. leprae laminin-binding protein predicts a 21-kDa molecule (ML-LBP21), which is smaller than the observed molecular size in SDS/PAGE. Immunofluorescence and immunoelectron microscopy on intact M. leprae with mAbs against recombinant (r) ML-LBP21 revealed that the protein is surface exposed. rML-LBP21 avidly bound to ␣2 laminins, the rG domain of the laminin-␣2 chain, and the native peripheral nerve laminin-2. The role of ML-LBP21 in Schwann cell adhesion and invasion was investigated by using fluorescent polystyrene beads coated with rML-LBP21. Although beads coated with rML-LBP21 alone specifically adhered to and were ingested by primary Schwann cells, these functions were significantly enhanced when beads were preincubated with exogenous ␣2 laminins. Taken together, the present data suggest that ML-LBP21 may function as a critical surface adhesin that facilitates the entry of M. leprae into Schwann cells.Nerve damage in leprosy results from Mycobacterium leprae invasion of Schwann cell of the peripheral nerves and is responsible for most of the deformity and disability of this disease (1, 2). Although patients can be cured of infection by multidrug therapy, the immunopathological sequelae responsible for the characteristic deformities of leprosy can continue during and even after antimicrobial therapy (3). Such therapeutic intervention has so far prevented only one third of infected individuals from suffering new disabilities (3). Therefore, interventions targeted directly to block the early interaction of M. leprae with peripheral nerve is important for the prevention of nerve infection and subsequent peripheral neuropathy.M. leprae invasion of Schwann cells of the peripheral nervous system represents an early crucial step leading to the nerve damage (1, 2). However, the surface molecules of M. leprae that mediate bacterial binding to and invasion of peripheral nerve are entirely unknown. As an important step toward identifying this invasion process, we have recently established that the laminin-2 isoform, specifically the G domain of laminin ␣2 chain, on the Schwann cell-axon unit serves as an initial neural target of M. leprae (4). Laminins, which consist of ␣, ␤, and ␥ chains, are major matrix proteins of basal laminae and play a crucial role in variety of cellular functions...