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.

Functional immune responses against SARS-CoV-2 variants of concern after fourth COVID-19 vaccine dose or infection in patients with blood cancer.

Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.

Disruption of the MSL complex inhibits tumour maintenance by exacerbating chromosomal instability.

Rewiring of cellular programmes in malignant cells generates cancer-specific vulnerabilities. Here, using an unbiased screening strategy aimed at identifying non-essential genes required by tumour cells to sustain unlimited proliferative capacity, we identify the male-specific lethal (MSL) acetyltransferase complex as a vulnerability of genetically unstable cancers. We find that disruption of the MSL complex and consequent loss of the associated H4K16ac mark do not substantially alter transcriptional programmes but compromise chromosome integrity and promote chromosomal instability (CIN) that progressively exhausts the proliferative potential of cancer cells through a p53-independent mechanism. This effect is dependent on pre-existing genomic instability, and normal cells are insensitive to MSL disruption. Using cell- and patient-derived xenografts from multiple cancer types, we show that excessive CIN induced by MSL disruption inhibits tumour maintenance. Our findings suggest that targeting MSL may be a valuable means to increase CIN beyond the level tolerated by cancer cells without inducing severe adverse effects in normal tissues.

Validation and Invalidation of Chemical Probes for the Human N-myristoyltransferases.

On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as target in cancer and infectious diseases. Here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis, and cell-cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 µM), D-NMAPPD (30 µM), or Tris-DBA palladium (10 µM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 µM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies that used off-target compounds.

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.

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.

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.

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.

Genome-wide, high-content siRNA screening identifies the Alzheimer's genetic risk factor FERMT2 as a major modulator of APP metabolism.

Genome-wide association studies (GWASs) have identified 19 susceptibility loci for Alzheimer's disease (AD). However, understanding how these genes are involved in the pathophysiology of AD is one of the main challenges of the "post-GWAS" era. At least 123 genes are located within the 19 susceptibility loci; hence, a conventional approach (studying the genes one by one) would not be time- and cost-effective. We therefore developed a genome-wide, high-content siRNA screening approach and used it to assess the functional impact of gene under-expression on APP metabolism. We found that 832 genes modulated APP metabolism. Eight of these genes were located within AD susceptibility loci. Only FERMT2 (a ß3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aß peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aß peptide production by raising levels of mature APP at the cell surface and facilitating its recycling. Taken as a whole, our data suggest that FERMT2 modulates the AD risk by regulating APP metabolism and Aß peptide production.

LppM impact on the colonization of macrophages by Mycobacterium tuberculosis.

Mycobacterium tuberculosis produces several bacterial effectors impacting the colonization of phagocytes. Here, we report that the putative lipoprotein LppM hinders phagocytosis by macrophages in a toll-like receptor 2-dependent manner. Moreover, recombinant LppM is able to functionally complement the phenotype of the mutant, when exogenously added during macrophage infection. LppM is also implicated in the phagosomal maturation, as a lppM deletion mutant is more easily addressed towards the acidified compartments of the macrophage than its isogenic parental strain. In addition, this mutant was affected in its ability to induce the secretion of pro-inflammatory chemokines, interferon-gamma-inducible protein-10, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α. Thus, our results describe a new mycobacterial protein involved in the early trafficking of the tubercle bacillus and its manipulation of the host immune response.

STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis is a successful intracellular pathogen. Numerous host innate immune responses signaling pathways are induced upon mycobacterium invasion, however their impact on M. tuberculosis replication is not fully understood. Here we reinvestigate the role of STAT3 specifically inside human macrophages shortly after M. tuberculosis uptake. We first show that STAT3 activation is mediated by IL-10 and occurs in M. tuberculosis infected cells as well as in bystander non-colonized cells. STAT3 activation results in the inhibition of IL-6, TNF-α, IFN-γ and MIP-1ß. We further demonstrate that STAT3 represses iNOS expression and NO synthesis. Accordingly, the inhibition of STAT3 is detrimental for M. tuberculosis intracellular replication. Our study thus points out STAT3 as a key host factor for M. tuberculosis intracellular establishment in the early stages of macrophage infection.

Testing chemical and genetic Modulators in Mycobacterium tuberculosis infected cells using phenotypic assays.

Mycobacterium tuberculosis is able to colonize host cells, and it is now well admitted that the intracellular stage of the bacteria contributes to tuberculosis pathogenesis as well as to making it a persistent infection. There is still limited understanding on how the tubercle bacillus colonizes the cell and what are the factors impacting on its intracellular persistence. Recent advances in imaging technique allow rapid quantification of biological objects in complex environments. Furthermore, M. tuberculosis is a microorganism that is particularly genetically tractable and that tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of M. tuberculosis expressing fluorescent proteins can be easily quantified by the use of confocal microscopy. Here we describe high-content/high-throughput imaging methods that enable tracking the bacillus inside host settings, taking into account the heterogeneity of colonization.