A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

Mycobacterium tuberculosis induces delayed lipid droplet accumulation in dendritic cells depending on bacterial viability and virulence.

Tuberculosis remains a global health threat with high morbidity. Dendritic cells (DCs) participate in the acute and chronic inflammatory responses to Mycobacterium tuberculosis (Mtb) by directing the adaptive immune response and are present in lung granulomas. In macrophages, the interaction of lipid droplets (LDs) with mycobacteria-containing phagosomes is central to host-pathogen interactions. However, the data available for DCs are still a matter of debate. Here, we reported that bone marrow-derived DCs (BMDCs) were susceptible to Mtb infection and replication at similar rate to macrophages. Unlike macrophages, the analysis of gene expression showed that Mtb infection induced a delayed increase in lipid droplet-related genes and proinflammatory response. Hence, LD accumulation has been observed by high-content imaging in late periods. Infection of BMDCs with killed H37Rv demonstrated that LD accumulation depends on Mtb viability. Moreover, infection with the attenuated strains H37Ra and Mycobacterium bovis-BCG induced only an early transient increase in LDs, whereas virulent Mtb also induced delayed LD accumulation. In addition, infection with the BCG strain with the reintroduced virulence RD1 locus induced higher LD accumulation and bacterial replication when compared to parental BCG. Collectively, our data suggest that delayed LD accumulation in DCs is dependent on mycobacterial viability and virulence.

The out-of-field dose in radiation therapy induces delayed tumorigenesis by senescence evasion.

A rare but severe complication of curative-intent radiation therapy is the induction of second primary cancers. These cancers preferentially develop not inside the planning target volume (PTV) but around, over several centimeters, after a latency period of 1-40 years. We show here that normal human or mouse dermal fibroblasts submitted to the out-of-field dose scattering at the margin of a PTV receiving a mimicked patient's treatment do not die but enter in a long-lived senescent state resulting from the accumulation of unrepaired DNA single-strand breaks, in the almost absence of double-strand breaks. Importantly, a few of these senescent cells systematically and spontaneously escape from the cell cycle arrest after a while to generate daughter cells harboring mutations and invasive capacities. These findings highlight single-strand break-induced senescence as the mechanism of second primary cancer initiation, with clinically relevant spatiotemporal specificities. Senescence being pharmacologically targetable, they open the avenue for second primary cancer prevention.

An original methodology to study polymeric nanoparticle-macrophage interactions: Nanoparticle tracking analysis in cell culture media and quantification of the internalized objects.

Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are among the most employed (co)polymers for the preparation of drug nanocarriers for the treatment of cancer and infectious diseases. Before considering any clinical use, it is necessary to understand the interactions between polymeric nanoparticles (NPs) and their physiological environment, especially immune cells. Here, we propose a simple, yet precise method to assess NPs internalization kinetics in macrophages, based on the direct analysis of the cell culture media after different incubation times. The proof of concept is given here by using fluorescent PLGA NPs. Nanoparticle tracking analysis (NTA) was a method of choice, enabling detecting each individual NP and analyzing its trajectory while in Brownian motion. As compared to dynamic light scattering (DLS), NTA enabled a more precise determination of NP size distribution. The uptake process was rapid: in one hour, around a third of the NPs were internalized. In addition, the internalized NPs were visualized by confocal microscopy. The fluorescent cellular stacks were analyzed using a freely available macro for ImageJ software, Particle_In_Cell-3D. The internalized objects were localized and counted. This methodology could serve for further studies while analyzing the effects of NPs size, shape and surface properties on their interaction with various cell lines.

High-Content Analysis Monitoring Intracellular Trafficking and Replication of Mycobacterium tuberculosis Inside Host Cells.

Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully understood and require further investigations. Therefore, increasing the knowledge of this process will help to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances in imaging techniques and images-based analysis allow the rapid quantification of biological objects in complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to track the bacillus within host cell settings.

Worms' Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms. In metazoans, they act as host defense factors by eliminating microbial pathogens. But they also help to select the colonizing bacterial symbionts while coping with specific environmental challenges. Although many AMPs share common structural characteristics, for example having an overall size between 10-100 amino acids, a net positive charge, a γ-core motif, or a high content of cysteines, they greatly differ in coding sequences as a consequence of multiple parallel evolution in the face of pathogens. The majority of AMPs is specific of certain taxa or even typifying species. This is especially the case of annelids (ringed worms). Even in regions with extreme environmental conditions (polar, hydrothermal, abyssal, polluted, etc.), worms have colonized all habitats on Earth and dominated in biomass most of them while co-occurring with a large number and variety of bacteria. This review surveys the different structures and functions of AMPs that have been so far encountered in annelids and nematodes. It highlights the wide diversity of AMP primary structures and their originality that presumably mimics the highly diverse life styles and ecology of worms. From the unique system that represents marine annelids, we have studied the effect of abiotic pressures on the selection of AMPs and demonstrated the promising sources of antibiotics that they could constitute.

Mitochondrial Dynamics and Activity in Legionella-Infected Cells.

The study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages.

Theaflavins, polyphenols of black tea, inhibit entry of hepatitis C virus in cell culture.

The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3'-monogallate (TF2), and theaflavin-3-3'-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.

Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis.

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.

Synergy between Circular Bacteriocin AS-48 and Ethambutol against Mycobacterium tuberculosis.

The increasing incidence of multidrug-resistant Mycobacterium tuberculosis strains and the very few drugs available for treatment are promoting the discovery and development of new molecules that could help in the control of this disease. Bacteriocin AS-48 is an antibacterial peptide produced by Enterococcus faecalis and is active against several Gram-positive bacteria. We have found that AS-48 was active against Mycobacterium tuberculosis, including H37Rv and other reference and clinical strains, and also against some nontuberculous clinical mycobacterial species. The combination of AS-48 with either lysozyme or ethambutol (commonly used in the treatment of drug-susceptible tuberculosis) increased the antituberculosis action of AS-48, showing a synergic interaction. Under these conditions, AS-48 exhibits a MIC close to some MICs of the first-line antituberculosis agents. The inhibitory activity of AS-48 and its synergistic combination with ethambutol were also observed on M. tuberculosis-infected macrophages. Finally, AS-48 did not show any cytotoxicity against THP-1, MHS, and J774.2 macrophage cell lines at concentrations close to its MIC. In summary, bacteriocin AS-48 has interesting antimycobacterial activity in vitro and low cytotoxicity, so further studies in vivo will contribute to its development as a potential additional drug for antituberculosis therapy.

Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors.

The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II.

Identification of Piperazinylbenzenesulfonamides as New Inhibitors of Claudin-1 Trafficking and Hepatitis C Virus Entry.

Hepatitis C virus (HCV) infection causes 500,000 deaths annually, in association with end-stage liver diseases. Investigations of the HCV life cycle have widened the knowledge of virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry of HCV into host cells is a complex process that is not fully understood but is characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines as well as primary hepatocytes. A functional characterization suggested a role for this compound and the compound SB399885, which share similar structures, as inhibitors of a late HCV entry step, modulating the localization of the coreceptor tight junction protein claudin-1 (CLDN1) in a 5-HT6-independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, an increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway, shared by several viruses, which involves G-protein-coupled receptor-dependent signaling in late steps of viral entry.IMPORTANCE The HCV entry process is highly complex, and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through the previously described activity of these inhibitors as antagonists of serotonin receptor 6 but instead through modulation of PKA activity in a 5-HT6-independent manner, as proven by the lack of 5-HT6 in the liver. We thus highlighted the involvement of the PKA pathway in modulating HCV entry at a postbinding step and in the recycling of the tight junction protein claudin-1 (CLDN1) toward the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for the PKA pathway as a regulator of CLDN1 recycling, with impacts on both cell biology and virology.

Proteomics of Mycobacterium Infection: Moving towards a Better Understanding of Pathogen-Driven Immunomodulation.

Intracellular bacteria are responsible for many infectious diseases in humans and have developed diverse mechanisms to interfere with host defense pathways. In particular, intracellular vacuoles are an essential niche used by pathogens to alter cellular and organelle functions, which facilitate replication and survival. Mycobacterium tuberculosis (Mtb), the pathogen causing tuberculosis in humans, is not only able to modulate its intraphagosomal fate by blocking phagosome maturation but has also evolved strategies to successfully prevent clearance by immune cells and to establish long-term survival in the host. Mass spectrometry (MS)-based proteomics allows the identification and quantitative analysis of complex protein mixtures and is increasingly employed to investigate host-pathogen interactions. Major challenges are limited availability and purity of pathogen-containing compartments as well as the asymmetric ratio in protein abundance when comparing bacterial and host proteins during the infection. Recent advances in purification techniques and MS technology helped to overcome previous difficulties and enable the detailed proteomic characterization of infected host cells and their pathogen-containing vacuoles. Here, we summarize current findings of the proteomic analysis of Mycobacterium-infected host cells and highlight progress that has been made to study the protein composition of mycobacterial vacuoles. Current investigations focus on the pathogenicity during Mtb infection, which will allow to better understand pathogen-induced changes and immunomodulation of infected host cells. Consequently, future research in this field will have important implications on host response, pathogen survival, and persistence, induced adaptive immunity and metabolic changes of immune cells promoting the development of novel host-directed therapies in tuberculosis.

Host-pathogen systems for early drug discovery against tuberculosis.

Tuberculosis (TB) is a global disease causing 1.8 million deaths each year. The appearance of drug-resistant strains raised the demand for new anti-mycobacterial drugs and therapies, because previously discovered antibiotics are shown to be inefficient. Moreover, the number of newly discovered drugs is not increasing in proportion to the emergence of drug resistance, which suggests that more optimized methodology and screening procedures are required including the incorporation of in vivo properties of TB infection. A way to improve efficacy of screening approaches is by introducing the use of different host-pathogen systems into primary screenings. These include whole cell-based screenings, zebrafish larvae-based screenings and the impact of artificial granuloma research on the drug discovery process. This review highlights current screening attempts and the identified molecular targets and summarizes findings of alternative, not fully explored host-pathogen systems for the characterization of anti-mycobacterial compounds.

Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids.

Mycobacterium tuberculosis is a major human pathogen that is able to survive inside host cells and resist immune clearance. Most particularly, it inhibits several arms of the innate immune response, including phagosome maturation or cytokine production. To better understand the molecular mechanisms by which M. tuberculosis circumvents host immune defenses, we used a transposon mutant library generated in a virulent clinical isolate of M. tuberculosis of the W/Beijing family to infect human macrophages, utilizing a cell line derivative of THP-1 cells expressing a reporter system for activation of the transcription factor NF-κB, a key regulator of innate immunity. We identified several M. tuberculosis mutants inducing a NF-κB activation stronger than that of the wild-type strain. One of these mutants was found to be deficient for the synthesis of cell envelope glycolipids, namely sulfoglycolipids, suggesting that the latter can interfere with innate immune responses. Using natural and synthetic molecular variants, we determined that sulfoglycolipids inhibit NF-κB activation and subsequent cytokine production or costimulatory molecule expression by acting as competitive antagonists of Toll-like receptor 2, thereby inhibiting the recognition of M. tuberculosis by this receptor. Our study reveals that producing glycolipid antagonists of pattern recognition receptors is a strategy used by M. tuberculosis to undermine innate immune defense. Sulfoglycolipids are major and specific lipids of M. tuberculosis, considered for decades as virulence factors of the bacilli. Our study uncovers a mechanism by which they may contribute to M. tuberculosis virulence.

Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages.

The intracellular bacteria Legionella pneumophila encodes a type IV secretion system (T4SS) that injects effector proteins into macrophages in order to establish and replicate within the Legionella-containing vacuole (LCV). Once generated, the LCV interacts with mitochondria through unclear mechanisms. We show that Legionella uses both T4SS-independent and T4SS-dependent mechanisms to respectively interact with mitochondria and induce mitochondrial fragmentation that ultimately alters mitochondrial metabolism. The T4SS effector MitF, a Ran GTPase activator, is required for fission of the mitochondrial network. These effects of MitF occur through accumulation of mitochondrial DNM1L, a GTPase critical for fission. Furthermore mitochondrial respiration is abruptly halted in a T4SS-dependent manner, while T4SS-independent upregulation of cellular glycolysis remains elevated. Collectively, these alterations in mitochondrial dynamics promote a Warburg-like phenotype in macrophages that favors bacterial replication. Hence the rewiring of cellular bioenergetics to create a replication permissive niche in host cells is a virulence strategy of L. pneumophila.

Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis.

A new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC 17 exhibited the best extracellular antitubercular activity (MIC50 = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC 17 may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.

Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis.

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis (Mtb) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv (MtbΔ33) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb, but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the MtbΔ33 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, MtbΔ33 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of MtbΔ33 with a frameshift allele also resulted in a Mtb strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of Mtb.

Host-directed therapies offer novel opportunities for the fight against tuberculosis.

Tuberculosis (TB) remains a leading global health problem that is exacerbated by the emergence of multidrug and extensively drug-resistant Mycobacterium tuberculosis strains. Control of the disease requires novel therapeutic strategies. Modulating host homeostasis appears to be a promising approach, and recent studies have identified novel potential host targets and compounds that could be investigated for host-directed therapies (HDTs). Moreover, the recent development of intracellular high-throughput phenotypic assays makes it possible to screen large libraries of compounds to identify more rapidly new effectors for mycobacterial elimination. Technological advances combined with the novel HDT concept opens an interesting and promising research area that could ultimately deliver personalized TB treatment.

Phenotypic assays for Mycobacterium tuberculosis infection.

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry.