Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i) binds the host complement regulators factor H and FHL-1 with high affinity, (ii) is the key molecule of the complement resistance of spirochetes, and (iii) is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.

Engagement of P2X7 on mouse dendritic cells, presumably by ATP released in response to contact allergen, is needed for IL-1β production and the sensitization phase of contact hypersensitivity.

Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of Aspergillus fumigatus. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of A. fumigatus in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (gliP⌬) and the the glip reconstituted strain (gliP R ). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliP⌬ strain was significantly less virulent than strain B-5233 or gliP R in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliP⌬, and gliP R strains showed the following: (i) deletion of gliP abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and gliP R , gliP⌬ CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the gliP R strain, but not the gliP⌬ strain. Our study confirms that gliotoxin is an important virulence determinant of A. fumigatus and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8+ T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA−/− or gzmB−/− mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, ΔΨm loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA−/− but not from gzmB−/− mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA−/− or gzmB−/− mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.