Dual-action compounds unleash a one-two punch against tuberculosis.

In this issue of Cell Chemical Biology, Gries et al.1 employ an innovative screening approach to identify anti-tuberculosis compounds with dual modes of action: anti-virulence against the type VII secretion system ESX-1 and enhanced ethionamide efficacy. These compounds hold promise for developing multi-target tuberculosis drugs with potential clinical applications.

ILC precursors differentiate into metabolically distinct ILC1-like cells during Mycobacterium tuberculosis infection.

Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces, and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis (Mtb) infection alters the phenotype and function of lung IL-18Rα+ ILC toward a protective interferon-γ-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program. Moreover, a BCG-driven type I milieu enhances the early generation of ILC1-like cells during secondary challenge with Mtb. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen-tailored immune response.

To Treat or Not to Treat Bees? Handy VarLoad: A Predictive Model for Varroa destructor Load.

The parasitic Varroa destructor is considered a major pathogenic threat to honey bees and to beekeeping. Without regular treatment against this mite, honey bee colonies can collapse within a 2-3-year period in temperate climates. Beyond this dramatic scenario, Varroa induces reductions in colony performance, which can have significant economic impacts for beekeepers. Unfortunately, until now, it has not been possible to predict the summer Varroa population size from its initial load in early spring. Here, we present models that use the Varroa load observed in the spring to predict the Varroa load one or three months later by using easily and quickly measurable data: phoretic Varroa load and capped brood cell numbers. Built on 1030 commercial colonies located in three regions in the south of France and sampled over a three-year period, these predictive models are tools designed to help professional beekeepers' decision making regarding treatments against Varroa. Using these models, beekeepers will either be able to evaluate the risks and benefits of treating against Varroa or to anticipate the reduction in colony performance due to the mite during the beekeeping season.

Preclinical assessment of a new live attenuated Mycobacterium tuberculosis Beijing-based vaccine for tuberculosis.

Tuberculosis still claims more lives than any other pathogen, and a vaccine better than BCG is urgently needed. One of the challenges for novel TB vaccines is to protect against all Mycobacterium tuberculosis lineages, including the most virulent ones, such as the Beijing lineage. Here we developed a live attenuated M. tuberculosis mutant derived from GC1237, a Beijing strain responsible for tuberculosis outbreaks in the Canary Islands. The mutant strain is inactivated both in the Rv1503c gene, responsible for surface glycolipid synthesis, and in the two-component global regulator PhoPR. This double mutant is as safe as BCG in immunodeficient SCID mice. In immune-competent mice and guinea pigs, the mutant is as protective as BCG against M. tuberculosis strains of common lineage 4 (Euro-American). By contrast, in mice the vaccine is protective against a M. tuberculosis strain of lineage 2 (East-Asian, Beijing), while BCG is not. These results highlight differences in protection efficacy of live attenuated M. tuberculosis-derived vaccine candidates depending on their genetic background, and provide insights for the development of novel live vaccines against TB, especially in East-Asian countries where M. tuberculosis strains of the Beijing family are highly dominant.

The Host Microbiota Contributes to Early Protection Against Lung Colonization by Mycobacterium tuberculosis.

Tuberculosis (TB), caused by the airborne bacterial pathogen Mycobacterium tuberculosis, remains a major source of morbidity and mortality worldwide. So far, the study of host-pathogen interactions in TB has mostly focused on the physiology and virulence of the pathogen, as well as, on the various innate and adaptive immune compartments of the host. Microbial organisms endogenous to our body, the so-called microbiota, interact not only with invading pathogens, but also with our immune system. Yet, the impact of the microbiota on host defense against M. tuberculosis remains poorly understood. In order to address this question, we adapted a robust and reproducible mouse model of microbial dysbiosis based on a combination of wide-spectrum antibiotics. We found that microbiota dysbiosis resulted in an increased early colonization of the lungs by M. tuberculosis during the first week of infection, correlating with an altered diversity of the gut microbiota during this time period. At the cellular level, no significant difference in the recruitment of conventional myeloid cells, including macrophages, dendritic cells and neutrophils, to the lungs could be detected during the first week of infection between microbiota-competent and -deficient mice. At the molecular level, microbiota depletion did not impact the global production of pro-inflammatory cytokines, such as interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)-1ß in the lungs. Strikingly, a reduced number of mucosal-associated invariant T (MAIT) cells, a population of innate-like lymphocytes whose development is known to depend on the host microbiota, was observed in the lungs of the antibiotics-treated animals after 1week of infection. These cells produced less IL-17A in antibiotics-treated mice. Notably, dysbiosis correction through the inoculation of a complex microbiota in antibiotics-treated animals reversed these phenotypes and improved the ability of MAIT cells to proliferate. Altogether, our results demonstrate that the host microbiota contributes to early protection of lung colonization by M. tuberculosis, possibly through sustaining the function(s) of MAIT cells. Our study calls for a better understanding of the impact of the microbiota on host-pathogen interactions in TB. Ultimately, this study may help to develop novel therapeutic approaches based on the use of beneficial microbes, or components thereof, to boost anti-mycobacterial immunity.

The role of the lung microbiota and the gut-lung axis in respiratory infectious diseases.

The pulmonary microbial community, described only a few years ago, forms a discreet part of the human host microbiota. The airway microbiota has been found to be substantially altered in the context of numerous respiratory disorders; nonetheless, its role in health and disease is as yet only poorly understood. Another important parameter to consider is the gut-lung axis, where distal (gut) immune modulation during respiratory disease is mediated by the gut microbiota. The use of specific microbiota strains, termed "probiotics," with beneficial effects on the host immunity and/or against pathogens, has proven successful in the treatment of intestinal disorders and is also showing promise in the context of airway diseases. In this review, we highlight the beneficial role of the body's commensal bacteria during airway infectious diseases, including recent evidence highlighting their local (lung) or distal (gut) contribution in this process.

Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

Assessment of the toxic effect of pesticides on honey bee drone fertility using laboratory and semifield approaches: A case study of fipronil.

Concern about the reproductive toxicity of plant protection products in honey bee reproducers is increasing. Because the reproductive capacity of honey bees is not currently considered during the risk assessment procedure performed during plant protection product registration, it is important to provide methods to assess such potential impairments. To achieve this aim, we used 2 different approaches that involved semifield and laboratory conditions to study the impact of fipronil on drone fertility. For each approach, the drones were reared for 20 d, from emergence to sexual maturity, and exposed to fipronil via a contaminated sugar solution. In both groups, the effects of fipronil were determined by studying life traits and fertility indicators. The results showed that the survival and maturity rates of the drones were better under laboratory conditions than under semifield conditions. Moreover, the drones reared under laboratory conditions produced more seminal fluid. Although these differences could be explained by environmental factors that may vary under semifield conditions, it was found that regardless of the approach used, fipronil did not affect survival rates, maturity rates, or semen volumes, whereas it did affect fertility by inducing a decrease in spermatozoa quantity that was associated with an increase in spermatozoa mortality. These results confirm that fipronil affects drone fertility and support the relevance of each approach for assessing the potential reproductive toxicity of plant protection products in honey bees. Environ Toxicol Chem 2017;36:2345-2351. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Wings as a new route of exposure to pesticides in the honey bee.

In pesticide risk assessment, estimating the routes and levels of exposure is critical. For honey bees subjected to pesticide spray, toxicity is assessed by thorax contact to account for all possible contact exposures. In the present study, the authors tested 6 active substances with different hydrophobicity. For the first time, the authors demonstrated that it is possible to induce mortality by pesticide contact with only the wings of the honey bee. The toxicities induced by contact with the wings and thorax were similar, with the wing median lethal dose (LD50) being 0.99 to 2.23 times higher than that of the thorax. This finding demonstrates that the wings represent a relevant route of exposure in the honey bee. In a second approach, the authors estimated the air volume displaced by the wings during 1 beating cycle to be 0.51 ± 0.03 cm(3), which corresponds to a volume of 116.8 ± 5.8 cm(3) s(-1) at a wing beat frequency of 230 Hz. The authors then tested realistic scenarios of exposure for bees flying through a pesticide cloud at different concentrations. In the worst-case scenario, the dose accumulated during the flight reached 525 ng bee(-1) s(-1). These results show that the procedure used to assess the risk posed by contact with pesticides could be improved by accounting for wing exposure.

A pragmatic approach to assess the exposure of the honey bee (Apis mellifera) when subjected to pesticide spray.

Plant protection spray treatments may expose non-target organisms to pesticides. In the pesticide registration procedure, the honey bee represents one of the non-target model species for which the risk posed by pesticides must be assessed on the basis of the hazard quotient (HQ). The HQ is defined as the ratio between environmental exposure and toxicity. For the honey bee, the HQ calculation is not consistent because it corresponds to the ratio between the pesticide field rate (in mass of pesticide/ha) and LD50 (in mass of pesticide/bee). Thus, in contrast to all other species, the HQ can only be interpreted empirically because it corresponds to a number of bees/ha. This type of HQ calculation is due to the difficulty in transforming pesticide field rates into doses to which bees are exposed. In this study, we used a pragmatic approach to determine the apparent exposure surface area of honey bees submitted to pesticide treatments by spraying with a Potter-type tower. The doses received by the bees were quantified by very efficient chemical analyses, which enabled us to determine an apparent surface area of 1.05 cm(2)/bee. The apparent surface area was used to calculate the exposure levels of bees submitted to pesticide sprays and then to revisit the HQ ratios with a calculation mode similar to that used for all other living species. X-tomography was used to assess the physical surface area of a bee, which was 3.27 cm(2)/bee, and showed that the apparent exposure surface was not overestimated. The control experiments showed that the toxicity induced by doses calculated with the exposure surface area was similar to that induced by treatments according to the European testing procedure. This new approach to measure risk is more accurate and could become a tool to aid the decision-making process in the risk assessment of pesticides.

Nitrogen metabolism in Mycobacterium tuberculosis physiology and virulence.

Several major pathogens, including Mycobacterium tuberculosis, parasitize host cells and exploit host-derived nutrients to sustain their own metabolism. Although the carbon sources that are used by M. tuberculosis have been extensively studied, the mechanisms by which mycobacteria capture and metabolize nitrogen, which is another essential constituent of biomolecules, have only recently been revisited. In this Progress article, we discuss central nitrogen metabolism in M. tuberculosis, the mechanisms that are used by this pathogen to obtain nitrogen from its host and the potential role of nitrogen capture and metabolism in virulence.

Amino acid capture and utilization within the Mycobacterium tuberculosis phagosome.

Mycobacterium tuberculosis, the agent of TB, is a facultative intracellular bacterial pathogen that replicates inside host macrophages and other phagocytes within a membrane-bound vacuole or phagosome. How M. tuberculosis captures and exploits vital nutrients inside host cells is an intensive research area that might lead to novel therapeutics for tuberculosis. Recent reports provided evidence that M. tuberculosis relies on amino acid uptake and degradation pathways to thrive inside its host. This opens novel research venues for the development of innovative antimicrobials against TB.

Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection.

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.

Mycobacterium tuberculosis nitrogen assimilation and host colonization require aspartate.

Here we identify the amino acid transporter AnsP1 as the unique aspartate importer in the human pathogen Mycobacterium tuberculosis. Metabolomic analysis of a mutant with an inactive AnsP1 revealed that the transporter is essential for M. tuberculosis to assimilate nitrogen from aspartate. Virulence of the AnsP1 mutant is impaired in vivo, revealing that aspartate is a primary nitrogen source required for host colonization by the tuberculosis bacillus.

Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages.

Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P(1)-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.

Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence.

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis-infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.

Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes.

Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane alpha helices and small ecto- and endodomains. A His6-tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-beta-d-maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins.