Neil E. Reiner

An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages

Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37°C ± pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-α. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.

The 19-kDa Mycobacterium tuberculosis Protein Induces Macrophage Apoptosis Through Toll-Like Receptor-2

Macrophages infected with Mycobacterium tuberculosis undergo increased rates of apoptosis. Important objectives are to define the microbial factors that cause apoptosis, the mechanisms involved and the impact on infection. The 19-kDa M. tuberculosis glycolipoprotein (p19) is both cell wall-associated and secreted and is a candidate virulence factor. We investigated the potential of recombinant, His-tagged p19 lacking the secretion/acylation signal to induce macrophage apoptosis. The TUNEL assay and annexin V binding to membrane phosphatidylserine were used to measure apoptosis. The results show that p19 does act to induce apoptosis in differentiated THP-1 cells and monocyte-derived macrophages and that this effect is both dose- and time-dependent. Furthermore, this effect of p19 is Toll-like receptor (TLR)-2-mediated because preincubation of either THP-1 cells or TLR-2-expressing CHO cells with anti-TLR-2 mAb inhibited apoptosis induced by p19. Apoptosis of macrophages in response to p19 was found to be caspase-8 dependent and caspase-9 independent consistent with a transmembrane pathway signaling cell death through TLR-2. The viability of M. tuberculosis in cells undergoing apoptosis induced by p19 was significantly reduced suggesting the possibility that this may favor containment of infection. Although native p19 is a mycobacterial glycolipoprotein, based upon the use of recombinant p19 where the acylation signal had been removed, we conclude that it is the polypeptide component of p19 that is responsible for signaling through TLR-2 and that the lipid moiety is not required.

Survival of Mycobacterium tuberculosis in Host Macrophages Involves Resistance to Apoptosis Dependent upon Induction of Antiapoptotic Bcl-2 Family Member Mcl-1

Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8–1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.

Proteomic analysis of the secretome of Leishmania donovani.

BackgroundLeishmania and other intracellular pathogens have evolved strategies that support invasion and persistence within host target cells. In some cases the underlying mechanisms involve the export of virulence factors into the host cell cytosol. Previous work from our laboratory identified one such candidate leishmania effector, namely elongation factor-1α, to be present in conditioned medium of infectious leishmania as well as within macrophage cytosol after infection. To investigate secretion of potential effectors more broadly, we used quantitative mass spectrometry to analyze the protein content of conditioned medium collected from cultures of stationary-phase promastigotes of Leishmania donovani, an agent of visceral leishmaniasis.ResultsAnalysis of leishmania conditioned medium resulted in the identification of 151 proteins apparently secreted by L. donovani. Ratios reflecting the relative amounts of each leishmania protein secreted, as compared to that remaining cell associated, revealed a hierarchy of protein secretion, with some proteins secreted to a greater extent than others. Comparison with an in silico approach defining proteins potentially exported along the classic eukaryotic secretion pathway suggested that few leishmania proteins are targeted for export using a classic eukaryotic amino-terminal secretion signal peptide. Unexpectedly, a large majority of known eukaryotic exosomal proteins was detected in leishmania conditioned medium, suggesting a vesicle-based secretion system.ConclusionThis analysis shows that protein secretion by L. donovani is a heterogeneous process that is unlikely to be determined by a classical amino-terminal secretion signal. As an alternative, L. donovani appears to use multiple nonclassical secretion pathways, including the release of exosome-like microvesicles.

The Salmonella spvB virulence gene encodes an enzyme that ADP‐ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells

ADP‐ribosylating enzymes, such as cholera and diphtheria toxins, are key virulence factors for a variety of extracellular bacterial pathogens but have not been implicated previously during intracellular pathogenesis. Salmonella strains are capable of invading epithelial cells and localizing in macrophages during infection. The spvB virulence gene of Salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. Here, we present evidence that spvB encodes an ADP‐ribosylating enzyme that uses actin as a substrate and depolymerizes actin filaments when expressed in CHO cells. Furthermore, site‐directed mutagenesis demonstrates that the ADP‐ribosylating activity of SpvB is essential for Salmonella virulence in mice. As spvB is expressed by Salmonella strains after invasion of epithelial cells or phagocytosis by macrophages, these results suggest that SpvB functions as an intracellular ADP‐ribosylating toxin critical for the pathogenesis of Salmonella infections.

1α,25-Dihydroxyvitamin D3-induced Monocyte Antimycobacterial Activity Is Regulated by Phosphatidylinositol 3-Kinase and Mediated by the NADPH-dependent Phagocyte Oxidase

We investigated the basis for the induction of monocyte antimycobacterial activity by 1α,25-dihydroxyvitamin D3 (D3). As expected, incubation of Mycobacterium tuberculosis-infected THP-1 cells or human peripheral blood, monocyte-derived macrophages with hormone resulted in the induction of antimycobacterial activity. This effect was significantly abrogated by pretreatment of cells with either of the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin or LY294002, or with antisense oligonucleotides to the p110 subunit of PI 3-Kα. Cells infected with M. tuberculosisalone or incubated with D3 alone produced little or undetectable amounts of superoxide anion (O⨪2). In contrast, exposure of M. tuberculosis-infected cells to D3 led to significant production of O⨪2, and this response was eliminated by either wortmannin, LY294002, or p110 antisense oligonucleotides. As was observed for PI 3-K inactivation, the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and degradative enzymes, polyethylene glycol coupled to either superoxide dismutase or catalase, also abrogated D3-induced antimycobacterial activity. Superoxide production by THP-1 cells in response to D3 required prior infection with liveM. tuberculosis, since exposure of cells to either killed M. tuberculosis or latex beads did not prime for an oxidative burst in response to subsequent hormone treatment. Consistent with these findings, redistribution of the cytosolic oxidase components p47 phox and p67 phox to the membrane fraction was observed in cells incubated with liveM. tuberculosis and D3 but not in response to combined treatment with heat-killed M. tuberculosis followed by D3. Redistribution of p47 phox and p67 phox to the membrane fraction in response to live M. tuberculosis and D3 was also abrogated under conditions where PI 3-K was inactivated. Taken together, these results indicate that D3-induced, human monocyte antimycobacterial activity is regulated by PI 3-K and mediated by the NADPH-dependent phagocyte oxidase.

Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles

The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.

Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine dephosphorylation and inhibition of mitogen-activated protein kinase in human mononuclear phagocytes. Role of the Src homology 2 containing tyrosine phosphatase 1.

Lipoarabinomannan (LAM) is a putative virulence factor of Mycobacterium tuberculosis that inhibits monocyte functions, and this may involve antagonism of cell signaling pathways. The effects of LAM on protein tyrosine phosphorylation in cells of the human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein kinase. To examine whether these effects of LAM could be related to activation of a phosphatase, fractions from LAM-treated cells were analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells, using phosphorylated mitogen-activated protein kinase as substrate, indicated that LAM promoted increased activity of SHP-1 in vivo. LAM also activated SHP-1 directly in vitro. Exposure of cells to LAM also attenuated the expression of tumor necrosis factor-α, interleukin-12, and major histocompatibility class II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.

Leishmania Exosomes Modulate Innate and Adaptive Immune Responses through Effects on Monocytes and Dendritic Cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ–producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100−/− L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100−/− leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100−/− exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

Altered cell signaling and mononuclear phagocyte deactivation during intracellular infection

Given the critical antimicrobial properties of mononuclear phagocytes, an important concern in cell biology and immunology has been to understand how intracellular microbes are able to establish states of chronic infection within these cells. Recent studies indicate that mononuclear phagocytes become functionally deactivated during intracellular infection. Here, Neil Reiner considers the experimental evidence to indicate that this is a frequent event that may be accounted for by induced defects in the signaling pathways required to bring cells to an activated state.