Summary To gain insight into the mechanisms by which host cells detect cytosolic invasion by intracellular pathogens, a genetic screen was performed to identify Listeria monocytogenes mutants that induced altered levels of host cell death. A mutation in lmo2473 resulted in hyper-stimulation of host cell death and IL-1β secretion (pyroptosis) following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 receptors, but dependent on ASC and AIM2. Importantly, wild type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. Since AIM2 is activated by DNA, these data suggested that pyroptosis is triggered by bacterial DNA released during lysis.
Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes , strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes . Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.
Recognition of the advantages of recombinant Listeria monocytogenes-based vaccines compared to those of other recombinant-vaccine platforms has facilitated the ongoing development and current evaluation of the former in early-phase clinical trials. These advantages include practical considerations, such as straightforward fermentation methods for manufacturing, and other desirable features, such as the ability to repeat administer even in the presence of protective L. monocytogenes-specific immunity (6,40,41). One compelling rationale for this vaccine platform is based on the well-known correlates of protection in the mouse listeriosis model: longlived functional CD4 ϩ and CD8 ϩ memory T cells induced in response to a single immunization with L. monocytogenes (19,28). There are now numerous publications that demonstrate the striking efficacy of recombinant L. monocytogenes vaccines in several animal models due to robust innate and adaptive cellular immunity (9,10,29). Recombinant L. monocytogenesbased vaccines represent an emerging approach to addressing an acute global need for effective vaccines that elicit functional cellular immunity to prevent or treat infections such as human immunodeficiency virus infection, hepatitis C virus infection, tuberculosis, and malaria as well as cancer.As L. monocytogenes is a food-borne pathogen having increased virulence among immunocompromised individuals, attenuated vaccine platforms are a prerequisite for advancement to evaluation with humans (23). We have previously described both live-attenuated and photochemically inactivated vaccine platforms derived from the wild-type (WT) strain 10403S (8, 9). The live-attenuated vaccine strain is deleted of both the actA and the inlB virulence genes (L. monocytogenes ⌬actA ⌬inlB vaccine strain), which in combination limit growth in the liver, a principal target organ of infection by the WT organism. Liver toxicity in mice, as measured by serum liver function tests for alanine transaminase and aspartate transaminase, is dramatically lower in mice injected intravenously (i.v.) with the L. monocytogenes ⌬actA ⌬inlB strain than in those injected i.v. with WT L. monocytogenes. Furthermore, liver toxicity was minimal and not dose limiting in two toxicology studies performed under good laboratory practice guidelines with cynomolgus monkeys given escalating doses of L. monocytogenes ⌬actA ⌬inlB-based strains (unpublished data). The L. monocytogenes ⌬actA ⌬inlB vaccine strain forms the basis for two ongoing FDA-approved phase 1 clinical trials being conducted with adult subjects with advanced cancers. The second vaccine platform, termed "killed but metabolically active" (KBMA), is derived from the L. monocytogenes ⌬actA ⌬inlB vaccine strain and is deleted of both uvrA and uvrB, genes encoding the DNA
SummaryA simple modi®cation to standard binary vector design has been utilized to enrich an Agrobacteriumtransformed population for plants containing only T-DNA sequences. A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors). The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the ef®ciency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.
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