Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum.

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-ß secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the ΔsecA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the ΔsecA2 strain. Therefore, to better examine secreted IFN-ß levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-ß to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-ß from macrophages infected with the ΔsecA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-ß response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.

Activation of host nucleic acid sensors by Mycobacterium: good for us or good for them?

Although the importance of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sensors in controlling viral infection is well established, their role in promoting an effective immune response to pathogens other than viruses is less clear. This is particularly true for infections with mycobacteria, as studies point to both protective and detrimental roles for activation of nucleic acid sensors in controlling a mycobacterial infection. Some of the contradiction likely stems from the use of different model systems and different mycobacterial species/strains as well as from which nucleic acid sensors were studied and what downstream effectors were evaluated. In this review, we will describe the different nucleic acid sensors that have been studied in the context of mycobacterial infections, and how the different studies compare. We conclude with a section on how nucleic acid sensor agonists have been used therapeutically and what further information is needed to enhance their potential as therapeutic agents.

Comparison of Ultrastructure, Extracellular Matrix, and Drug Susceptibility in M. avium subs. hominissuis Biofilms.

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and the drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four-week incubation in M63 medium or 24 h exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in the biofilm ultrastructure between the two methods, including variation in the appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring the susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at greater than 10-fold lower rates than planktonic cells at almost all concentrations. These comparisons indicate differences in biofilm responses by M. avium under differing conditions, but also suggest common features of biofilm formation, including cellulose production and antimicrobial resistance.

Tumor-Derived Extracellular Vesicles: A Means of Co-opting Macrophage Polarization in the Tumor Microenvironment.

Extracellular vesicles (EVs) are a heterogeneous population of membrane-bound parcels of bioactive proteins, nucleic acids, and lipids released from almost all cell types. The diversity of cargo packaged into EVs proffer the induction of an array of effects on recipient cells. EVs released from tumor cells have emerged as a vital means of communication and immune modulation within the tumor microenvironment (TME). Macrophages are an important contributor to the TME with seemingly paradoxical roles promoting either pro- or anti-tumoral immune function depending on their activated phenotypes. Here, we discuss the influence of tumor-derived extracellular vesicles on the functional plasticity of macrophages in tumor progression.

Bacteria- and host-derived extracellular vesicles - two sides of the same coin?

Intracellular bacterial pathogens spend portions of their life cycle both inside and outside host cells. While in these two distinct environments, they release or shed bacterial components, including virulence factors that promote their survival and replication. Some of these components are released through extracellular vesicles, which are either derived from the bacteria themselves or from the host cells. Bacteria- and host-derived vesicles have been studied almost exclusively in isolation from each other, with little discussion of the other type of secreted vesicles, despite the fact that both are generated during an in vivo infection and both are likely play a role in bacterial pathogenesis and host immunity. In this Review, we aim to bridge this gap and discuss what we know of bacterial membrane vesicles in their generation and composition. We will compare and contrast this with the composition of host-derived vesicles with regard to bacterial components. We will also compare host cell responses to the different vesicles, with a focus on how these vesicles modulate the immune response, using Mycobacterium, Listeria and Salmonella as specific examples for these comparisons.

Host cytosolic RNA sensing pathway promotes T Lymphocyte-mediated mycobacterial killing in macrophages.

Mycobacterial infection leads to activation of the RIG-I/MAVS/TBK1 RNA sensing pathway in macrophages but the consequences of this activation remains poorly defined. In this study, we determined that activation of this RNA sensing pathway stimulates ICAM-1 expression in M.avium-infected macrophage through the inhibition of the E3 ubiquitin ligase CRL4COP1/DET1. CRL4 when active targets the transcription factor ETV5 for degradation by the ubiquitin-proteasome system. In the absence of the ETV5 transcription factor, ICAM-1 expression is significantly decreased. The M.avium-induced ICAM-1 production is required for the formation of immune synapse between infected macrophages and antigen-specific CD4+ T lymphocytes, and is essential for CD4+ T lymphocyte-mediated mycobacterial killing in vitro and in mice. This study demonstrates a previously undefined mechanism by which a host cytosolic RNA sensing pathway contributes to the interplay between mycobacteria infected macrophages and antigen-specific T lymphocytes.

Mycobacterium tuberculosis-induced IFN-ß production requires cytosolic DNA and RNA sensing pathways.

RNA sensing pathways are key elements in a host immune response to viral pathogens, but little is known of their importance during bacterial infections. We found that Mycobacterium tuberculosis (M.tb) actively releases RNA into the macrophage cytosol using the mycobacterial SecA2 and ESX-1 secretion systems. The cytosolic M.tb RNA induces IFN-ß production through the host RIG-I/MAVS/IRF7 RNA sensing pathway. The inducible expression of IRF7 within infected cells requires an autocrine signaling through IFN-ß and its receptor, and this early IFN-ß production is dependent on STING and IRF3 activation. M.tb infection studies using Mavs-/- mice support a role for RNA sensors in regulating IFN-ß production and bacterial replication in vivo. Together, our data indicate that M.tb RNA is actively released during an infection and promotes IFN-ß production through a regulatory mechanism involving cross-talk between DNA and RNA sensor pathways, and our data support the hypothesis that bacterial RNA can drive a host immune response.

Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice.

Endothelial cells play an essential role in regulating an immune response through promoting leukocyte adhesion and cell migration and production of cytokines such as TNFα. Regulation of endothelial cell immune function is tightly regulated and recent studies suggest that extracellular vesicles (EVs) are prominently involved in this process. However, the importance of EVs in regulating endothelial activation in the context of a bacterial infection is poorly understood. To begin addressing this knowledge gap we characterized the endothelial cell response to EVs released from Mycobacterium tuberculosis (Mtb) infected macrophages. Our result showed increased macrophage migration through the monolayer when endothelial cells were pretreated with EVs isolated from Mtb-infected macrophages. Transcriptome analysis showed a significant upregulation of genes involved in cell adhesion and the inflammatory process in endothelial cells treated with EVs. These results were validated by quantitative PCR and flow cytometry. Pathway analysis of these differentially expressed genes indicated that several immune response-related pathways were up-regulated. Endothelial cells were also treated with EVs isolated from the serum of Mtb-infected mice. Interestingly, EVs isolated 14 days but not 7 or 21 days post-infection showed a similar ability to induce endothelial cell activation suggesting a change in EV function during the course of an Mtb infection. Immunofluorescence microscopy result indicated that NF-κB and the Type 1 interferon pathways were involved in endothelial activation by EVs. In summary, our data suggest that EVs can activate endothelial cells and thus may play an important role in modulating host immune responses during an Mtb infection.

Regulation and mechanisms of extracellular vesicle biogenesis and secretion.

EV (extracellular vesicle) biology is a rapidly expanding field. These heterogeneous membrane vesicles, which are shed from virtually all cell types, collectively represent a new dimension of intercellular communication in normal physiology and disease. They have been shown to deliver infectious and pathogenic agents to non-infected cells whereas in cancers they are thought to condition the tumor microenvironment. Their presence in body fluids and inherent capacity for systemic delivery point to their clinical promise. All of the above only intensifies the need to better understand the classification, mode of biogenesis, and contents of the different subtypes of EVs. This article focusses on vesicle subtypes labeled as exosomes and MVs (microvesicles) and discusses the biogenesis and release of these vesicles from cells.

Protein Kinase Inhibitors as Potential Antimicrobial Drugs Against Tuberculosis, Malaria and HIV.

Infectious diseases that are caused by pathogenic microbes such as bacteria, viruses, parasites or fungi remain the top major cause of death across the world, particularly in low income countries, and may be transmitted from person to person, or from insects or animals. In general, infectious diseases may be treated with antimicrobial agents including antibiotics, antiviral, antifungal or antiparasitic medications. The therapeutic application of antimicrobial drugs in the 20th century substantially contributed to the global control of infectious diseases worldwide. However, pathogenic microbes have evolved various mechanisms to render the antimicrobial drugs less effective. This has resulted in an increasing number of people infected with pathogenic microbes that are resistant to antimicrobial drugs, and in some cases leading to untreatable infections. Therefore, new antimicrobial drugs are urgently needed to prevent possible recurrence and emergence of previously treatable infectious diseases. In the past decades, protein kinase inhibitors have become an attractive area in the development of novel antimicrobial drugs. In the current review, we will describe the recent efforts in the development of microbial and host protein kinase-targeting inhibitors as potential antimicrobial drugs against HIV, tuberculosis and malaria.

Exosomes function in antigen presentation during an in vivo Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis-infected macrophages and dendritic cells are limited in their ability to present antigen to CD4+ T cells suggesting that other mechanism of antigen presentation are driving the robust T cell response observed during an M. tuberculosis infection. These mechanisms could include antigens present in apoptotic bodies, necrotic debris, exosomes or even release of non-vesicular antigen from infected cells. However, there is limited data to support any of these mechanisms as important in driving T cell activation in vivo. In the present study we use Rab27a-deficient mice which show diminished trafficking of mycobacterial components to exosomes as well as M. tuberculosis strains that express recombinant proteins which traffic or fail to traffic to exosomes. We observed that exosomes released during a mouse M. tuberculosis infection contribute significantly to its T cell response. These finding imply that exosomes function to promote T cell immunity during a bacterial infection and are an important source of extracellular antigen.

Extracellular vesicles and infectious diseases: new complexity to an old story.

Exosomes and other extracellular microvesicles (ExMVs) have important functions in intercellular communication and regulation. During the course of infection, these vesicles can convey pathogen molecules that serve as antigens or agonists of innate immune receptors to induce host defense and immunity, or that serve as regulators of host defense and mediators of immune evasion. These molecules may include proteins, nucleic acids, lipids, and carbohydrates. Pathogen molecules may be disseminated by incorporation into vesicles that are created and shed by host cells, or they may be incorporated into vesicles shed from microbial cells. Involvement of ExMVs in the induction of immunity and host defense is widespread among many pathogens, whereas their involvement in immune evasion mechanisms is prominent among pathogens that establish chronic infection and is found in some that cause acute infection. Because of their immunogenicity and enrichment of pathogen molecules, exosomes may also have potential in vaccine preparations and as diagnostic markers. Additionally, the ability of exosomes to deliver molecules to recipient cells raises the possibility of their use for drug/therapy delivery. Thus, ExMVs play a major role in the pathogenesis of infection and provide exciting potential for the development of novel diagnostic and therapeutic approaches.

Innate Immune Responses to Tuberculosis.

Tuberculosis remains one of the greatest threats to human health. The causative bacterium, Mycobacterium tuberculosis, is acquired by the respiratory route. It is exquisitely adapted to humans and is a prototypic intracellular pathogen of macrophages, with alveolar macrophages being the primary conduit of infection and disease. However, M. tuberculosis bacilli interact with and are affected by several soluble and cellular components of the innate immune system which dictate the outcome of primary infection, most commonly a latently infected healthy human host, in whom the bacteria are held in check by the host immune response within the confines of tissue granuloma, the host histopathologic hallmark. Such individuals can develop active TB later in life with impairment in the immune system. In contrast, in a minority of infected individuals, the early host immune response fails to control bacterial growth, and progressive granulomatous disease develops, facilitating spread of the bacilli via infectious aerosols. The molecular details of the M. tuberculosis-host innate immune system interaction continue to be elucidated, particularly those occurring within the lung. However, it is clear that a number of complex processes are involved at the different stages of infection that may benefit either the bacterium or the host. In this article, we describe a contemporary view of the molecular events underlying the interaction between M. tuberculosis and a variety of cellular and soluble components and processes of the innate immune system.

Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes.

Exosomes are extracellular vesicles of endocytic origin that function in intercellular communication. Our previous studies indicate that exosomes released from Mycobacterium tuberculosis-infected macrophages contain soluble mycobacterial proteins. However, it was unclear how these secreted proteins were targeted to exosomes. In this study, we determined that exosome production by the murine macrophage cell line RAW264.7 requires the endosomal sorting complexes required for transport and that trafficking of mycobacterial proteins from phagocytosed bacilli to exosomes was dependent on protein ubiquitination. Moreover, soluble mycobacterial proteins, when added exogenously to RAW264.7 or human HEK293 cells, were endocytosed, ubiquitinated, and released via exosomes. This suggested that endocytosed proteins could be recycled from cells through exosomes. This hypothesis was supported using the tumor-associated protein He4, which, when endocytosed by RAW264.7 or HEK293 cells, was transported to exosomes in a ubiquitin-dependent manner. Our data suggest that ubiquitination is a modification sufficient for trafficking soluble proteins within the phagocytic/endocytic network to exosomes.

Exosomal RNA from Mycobacterium tuberculosis-Infected Cells Is Functional in Recipient Macrophages.

Exosomes are extracellular vesicles released by cells that carry proteins, lipids and nucleic acids and function in intercellular communication. Previously, we determined that exosomes released from Mycobacterium tuberculosis (M.tb)-infected macrophages carry mycobacterial proteins and lipids. However, the RNA composition within these exosomes has not been defined. In this study, we characterized the exosomes released from M.tb-infected macrophages and identified a cohort of mouse messenger RNA (mRNA) and microRNA (miRNA). Quantitative reverse-transcriptase polymerase chain reaction analysis showed less abundance of miRNAs in exosomes released from infected compared with uninfected macrophages. Moreover, more than 100 transcripts were found to be enriched or unique to exosomes from infected cells including transcripts involved in regulating an immune response. The exosomal RNA could be transferred and expressed in naïve macrophages and was biologically active, stimulating production of inflammatory mediators and inducing apoptosis in recipient cells. Interestingly, we also identified mycobacterial transcripts in exosomes released from infected macrophages. To our knowledge, this is the first study to identify bacterial-derived RNA in exosomes. Our results suggest that exosomal RNA released from M.tb-infected macrophages may have functional and diagnostic potential in the context of a mycobacterial infection.

Exosomes and other extracellular vesicles in host-pathogen interactions.

An effective immune response requires the engagement of host receptors by pathogen-derived molecules and the stimulation of an appropriate cellular response. Therefore, a crucial factor in our ability to control an infection is the accessibility of our immune cells to the foreign material. Exosomes-which are extracellular vesicles that function in intercellular communication-may play a key role in the dissemination of pathogen- as well as host-derived molecules during infection. In this review, we highlight the composition and function of exosomes and other extracellular vesicles produced during viral, parasitic, fungal and bacterial infections and describe how these vesicles could function to either promote or inhibit host immunity.

Bactericidal activity of an imidazo[1, 2-a]pyridine using a mouse M. tuberculosis infection model.

Tuberculosis remains a global threat due in part to the long treatment regimen and the increased prevalence of drug resistant M. tuberculosis strains. Therefore, new drug regimens are urgently required to combat this deadly disease. We previously synthesized and evaluated a series of new anti-tuberculosis compounds which belong to the family of imidazo[1,2-a]pyridines. This family of compounds showed low nM MIC (minimal inhibitory concentration) values against M. tuberculosis in vitro. In this study, a derivative of imidazo[1,2-a]pyridines, (N-(4-(4-chlorophenoxy)benzyl)-2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamide) (ND-09759), was selected as a promising lead compound to determine its protective efficacy using a mouse infection model. Pharmacokinetic analysis of ND-09759 determined that at a dosage of 30 mg/kg mouse body weight (PO) gave a maximum serum drug concentration (Cmax) of 2.9 µg/ml and a half-life of 20.1 h. M. tuberculosis burden in the lungs and spleens was significantly decreased in mice treated once daily 6 days per week for 4-weeks with ND-09759 compared to untreated mice and this antibiotic activity was equivalent to isoniazid (INH) and rifampicin (RMP), two first-line anti-TB drugs. We observed slightly higher efficacy when using a combination of ND-09759 with either INH or RMP. Finally, the histopathological analysis revealed that infected mice treated with ND-09759 had significantly reduced inflammation relative to untreated mice. In conclusion, our findings indicate ND-09759 might be a potent candidate for the treatment of active TB in combination with current standard anti-TB drugs.

Mannose receptor-dependent delay in phagosome maturation by Mycobacterium avium glycopeptidolipids.

The ability of pathogenic mycobacteria to block phagosome-lysosome fusion is critical for its pathogenesis. The molecules expressed by mycobacteria that inhibit phagosome maturation and the mechanism of this inhibition have been extensively studied. Recent work has indicated that mannosylated lipoarabinomannan (ManLAM) isolated from Mycobacterium tuberculosis can function to delay phagosome-lysosome fusion and that this delay requires the interaction of ManLAM with the mannose receptor (MR). However, the molecules expressed by other pathogenic mycobacteria that function to inhibit phagosome maturation have not been well described. In the present study, we show that phagosomes containing silica beads coated with glycopeptidolipids (GPLs), a major surface component of Mycobacterium avium, showed limited acidification and delayed recruitment of late endosomal/lysosomal markers compared to those of phosphatidylcholine-coated beads. The carbohydrate component of the GPLs was required, as beads coated only with the lipopeptide core failed to delay phagosome-lysosome fusion. Moreover, the ability of GPLs to delay phagosome maturation was dependent on the macrophage expression of the MR. Using CHO cells expressing the MR, we confirmed that the GPLs bind this receptor. Finally, human monocyte-derived macrophages knocked down for MR expression showed increased M. avium phagosome-lysosome fusion relative to control cells. Together, the data indicate that GPLs can function to delay phagosome-lysosome fusion and suggest that GPLs, like ManLAM, work through the MR to mediate this activity.

Toll-like receptor signaling in hen ovarian granulosa cells is dependent on stage of follicle maturation.

The recent identification of toll-like receptor (TLR) signaling within ovarian granulosa cells has broad implications for ovarian physiology. Functions of TLRs within granulosa cells of the laying hen are of particular interest due to the method of transovarian transmission of Salmonella enteritidis, which results in egg contamination. This study utilized hen granulosa cells to evaluate the expression and function of Gallus TLR-signaling at distinct stages of follicular maturity. Data presented herein demonstrate the presence of TLR2, TLR4, and TLR15 mRNAs in undifferentiated granulosa cells from prehierarchal follicles and differentiated granulosa cells from preovulatory follicles, together with mRNAs encoding adaptor proteins and signaling components required for TLR signaling gene. Treatment with lipopolysaccharide (LPS) or LH, in vitro, led to the differential regulation of TLRs based on the stage of follicle maturation, with the largest (F1) follicle granulosa cells having the most rapid response. Furthermore, treatment with LPS resulted in attenuation of agonist-induced progesterone synthesis in undifferentiated, but not differentiated, granulosa cells. Additionally, undifferentiated granulosa cells were significantly more sensitive to LPS-induced apoptosis than differentiated granulosa cells from the F1 follicle. Together, these data provide evidence for a complete and functional TLR signaling pathway in hen granulosa cells, with effects on steroidogenesis and cell viability dependent upon stage of maturation. These differences may reflect the susceptibility of granulosa cells at early stages of maturation to undergo apoptosis in response to select pathogenic stimuli, thus attenuating transovarian transmission, whereas granulosa cells from preovulatory follicles are comparably resistant to LPS-mediated apoptosis.

ADP-ribosylation factor 6 regulates tumorigenic and invasive properties in vivo.

This study shows that the small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is an important regulator of tumor growth and metastasis. Using spontaneous melanoma tumor growth assays and experimental metastasis assays in nude mice, we show that sustained activation of ARF6 reduces tumor mass growth but significantly enhances the invasive capacity of tumor cells. In contrast, mice injected with tumor cells expressing a dominantly inhibitory ARF6 mutant exhibited a lower incidence and degree of invasion and lung metastasis compared with control animals. Effects on tumor growth correlate with reduced cell proliferation capacity and are linked at least in part to alterations in mitotic progression induced by defective ARF6 cycling. Furthermore, phospho-ERK levels in subcultured cells from ARF6(GTP) and ARF6(GDP) tumor explants correlate with invasive capacity. ARF6-induced extracellular signal-regulated kinase (ERK) signaling leads to Rac1 activation to promote invadopodia formation and cell invasion. These findings document an intricate role for ARF6 and the regulation of ERK activation in orchestrating mechanisms underlying melanoma growth, invasion, and metastases.