Toxoplasma gondii Inhibits Covalent Modification of Histone H3 at the IL-10 Promoter in Infected Macrophages

Infection of macrophages with the protozoan parasite Toxoplasma gondii results in inhibition of a large panel of LPS-responsive cytokines, including TNF-α, while leaving others such as IL-10 intact. Recent studies provide evidence that the parasite interferes with chromatin remodeling at the TNF-α promoter that is normally associated with LPS stimulation, but that is not required for TLR4 induction of IL-10. Here, we examined the effect of Toxoplasma on IL-10 induced by simultaneous signaling through TLR4 and FcγR, a combined stimulus that triggers histone H3 covalent modification at the IL-10 promoter resulting in high level IL-10 cytokine production. We show that the parasite inhibits high level IL-10 production and prevents histone H3 Ser10 phosphorylation and Lys9/14 acetylation at the IL-10 promoter. These results provide compelling evidence that T. gondii targets the host cell chromatin remodeling machinery to down-regulate cytokine responses in infected macrophages

Mycoplasma Suppression of THP-1 Cell TLR Responses Is Corrected with Antibiotics

Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 β was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1β and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma

HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

Background Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. Methodology/Principal Findings We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. Conclusions/Significance The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death

REUL Is a Novel E3 Ubiquitin Ligase and Stimulator of Retinoic-Acid-Inducible Gene-I

RIG-I and MDA5 are cytoplasmic sensors that recognize different species of viral RNAs, leads to activation of the transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons. In this study, we identified REUL, a RING-finger protein, as a specific RIG-I-interacting protein. REUL was associated with RIG-I, but not MDA5, through its PRY and SPRY domains. Overexpression of REUL potently potentiated RIG-I-, but not MDA5-mediated downstream signalling and antiviral activity. In contrast, the RING domain deletion mutant of REUL suppressed Sendai virus (SV)-induced, but not cytoplasmic polyI:C-induced activation of IFN-β promoter. Knockdown of endogenous REUL by RNAi inhibited SV-triggered IFN-β expression, and also increased VSV replication. Full-length RIG-I, but not the CARD domain deletion mutant of RIG-I, underwent ubiquitination induced by REUL. The Lys 154, 164, and 172 residues of the RIG-I CARD domain were critical for efficient REUL-mediated ubiquitination, as well as the ability of RIG-I to induce activation of IFN-β promoter. These findings suggest that REUL is an E3 ubiquitin ligase of RIG-I and specifically stimulates RIG-I-mediated innate antiviral activity

Immune Response to Lactobacillus plantarum Expressing Borrelia burgdorferi OspA Is Modulated by the Lipid Modification of the Antigen

Over the past decade there has been increasing interest in the use of lactic acid bacteria as mucosal delivery vehicles for vaccine antigens, microbicides and therapeutics. We investigated the mechanism by which a mucosal vaccine based in recombinant lactic acid bacteria breaks the immunological tolerance of the gut in order to elicit a protective immune response.We analyzed how the lipid modification of OspA affects the localization of the antigen in our delivery vehicle using a number of biochemistry techniques. Furthermore, we examined how OspA-expressing L. plantarum breaks the oral tolerance of the gut by stimulating human intestinal epithelial cells, peripheral blood mononuclear cells and monocyte derived dendritic cells and measuring cytokine production. We show that the leader peptide of OspA targets the protein to the cell envelope of L. plantarum, and it is responsible for protein export across the membrane. Mutation of the lipidation site in OspA redirects protein localization within the cell envelope. Further, we show that lipidated-OspA-expressing L. plantarum does not induce secretion of the pro-inflammatory cytokine IL-8 by intestinal epithelial cells. In addition, it breaks oral tolerance of the gut via Th1/Th2 cell mediated immunity, as shown by the production of pro- and anti-inflammatory cytokines by human dendritic cells, and by the production of IgG2a and IgG1 antibodies, respectively.Lipid modification of OspA expressed in L. plantarum modulates the immune response to this antigen through a Th1/Th2 immune response

Multiple M. tuberculosis Phenotypes in Mouse and Guinea Pig Lung Tissue Revealed by a Dual-Staining Approach

A unique hallmark of tuberculosis is the granulomatous lesions formed in the lung. Granulomas can be heterogeneous in nature and can develop a necrotic, hypoxic core which is surrounded by an acellular, fibrotic rim. Studying bacilli in this in vivo microenvironment is problematic as Mycobacterium tuberculosis can change its phenotype and also become acid-fast negative. Under in vitro models of differing environments, M. tuberculosis alters its metabolism, transcriptional profile and rate of replication. In this study, we investigated whether these phenotypic adaptations of M. tuberculosis are unique for certain environmental conditions and if they could therefore be used as differential markers. Bacilli were studied using fluorescent acid-fast auramine-rhodamine targeting the mycolic acid containing cell wall, and immunofluorescence targeting bacterial proteins using an anti-M. tuberculosis whole cell lysate polyclonal antibody. These techniques were combined and simultaneously applied to M. tuberculosis in vitro culture samples and to lung sections of M. tuberculosis infected mice and guinea pigs. Two phenotypically different subpopulations of M. tuberculosis were found in stationary culture whilst three subpopulations were found in hypoxic culture and in lung sections. Bacilli were either exclusively acid-fast positive, exclusively immunofluorescent positive or acid-fast and immunofluorescent positive. These results suggest that M. tuberculosis exists as multiple populations in most conditions, even within seemingly a single microenvironment. This is relevant information for approaches that study bacillary characteristics in pooled samples (using lipidomics and proteomics) as well as in M. tuberculosis drug development

How is the phagocyte lectin keyboard played? Master class lesson by Mycobacterium tuberculosis

International audienceMammals have evolved surface pattern recognition receptors, such as the Toll-like receptors, to initiate defenses against pathogens, including mycobacterium. In turn, microbes have developed strategies to circumvent defenses of their host and establish persistent infections. Mycobacterium tuberculosis, one of the most successful pathogens worldwide, has the ability to parasitize and manipulate phagocytic cells of its human host. A set of recent reports has shed light on exploitation of phagocyte surface lectins by the tubercle bacillus. These findings could lead the way to innovative therapeutic approaches

Signature-Tagged Transposon Mutagenesis Identifies Novel Mycobacterium tuberculosis Genes Involved in the Parasitism of Human Macrophages

Using signature-tagged transposon mutagenesis, we isolated 23 Mycobacterium tuberculosis mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells

A murine DC-SIGN homologue contributes to early host defense against Mycobacterium tuberculosis

International audienceThe C-type lectin dendritic cell−specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen