Yannick Poquet

Mycobacterial P1-Type ATPases Mediate Resistance to Zinc Poisoning in Human Macrophages

Summary 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 P1-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.

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 inactivated in AnsP1 revealed the transporter is essential for M. tuberculosis to assimilate nitrogen from aspartate. Virulence of the AnsP1 mutant is impaired in vivo, revealing aspartate is a primary nitrogen source required for host colonization by the tuberculosis bacillus.

The spatial distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus varies during the cell cycle.

Recently, we have developed a photocrosslinking approach which uses anthracene as a photoactivatable group and which allows us to determine the lateral distribution of lipids in membranes quantitatively. In synchronous cultures of the gram‐positive bacterium Micrococcus luteus, this approach shows that the spatial distribution of phosphatidylglycerol and diamnnosyldiacylglycerol, the two major lipids in the bacterial membrane, varies greatly during the cell cycle. Minimum heterogeneity was observed during cell growth while maximum heterogeneity was detected during cell division.

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.

A central role for aspartate in Mycobacterium tuberculosis physiology and virulence

The tuberculosis (TB) bacillus, Mycobacterium tuberculosis, is a facultative intracellular pathogen that multiplies inside macrophages, in which it resides within a specialized compartment, the phagosome, where nutrient sources are likely limited. A number of studies provided compelling evidence that M. tuberculosis has the ability to exploit host-derived carbon sources, such as triglycerides, cholesterol and glucose, and to proliferate inside host cells (Mckinney et al., 2000; Pandey and Sassetti, 2008; Daniel et al., 2011; Marrero et al., 2013). In addition to carbon, nitrogen is an essential constituent of all living organisms. As compared to carbon requirements, little is known about the nature of nitrogen-containing molecules utilized by the TB bacillus during infection. We recently discovered that nitrogen incorporation from exogenous aspartate is required for host colonization by M. tuberculosis (Gouzy et al., 2013). This study highlights, for the first time, the potential of amino acids, and aspartate in particular, as a major nitrogen reservoir supporting M. tuberculosis virulence in vivo. It also opens a series of questions to be addressed in the future. Chief among these questions is the origin and molecular mechanism(s) of transport allowing mycobacterial access to aspartate inside host cells. TB lesions are enriched in aspartate, and this likely relies on global metabolic changes in immune cells during granuloma formation (Somashekar et al., 2011). How nutrients, such as aspartate, access the M. tuberculosis phagosome during infection is an intriguing issue. Using mass spectrometry imaging, we further showed that aspartate can access the mycobacterial phagosome, at least in vitro (Gouzy et al., 2013). In mammalian cells, aspartate uptake relies on transporters of the solute carriers (SLC) superfamily, and in particular on those of the high-affinity glutamate and neutral amino acid (SLC1) and of the cationic amino acid (SLC7) transporter families. Among these transporters, SLC1A2 was reported to mediate aspartate transport inside macrophages (Rimaniol et al., 2001; Ye et al., 2010). Interestingly, we reported the expression of the slc1a2 gene is increased in human macrophages upon M. tuberculosis infection (Tailleux et al., 2008). Increased expression of this transporter could impact considerably the ability of M. tuberculosis to multiply inside macrophages, as it is the case for the neutral amino acid transporter SLC1A5 in the context of Francisella tularensis and Legionella pneumophila infection (Wieland et al., 2005; Barel et al., 2012). These studies suggested the transport of yet to be identified amino acid(s) is important to sustain intracellular multiplication of these two bacterial species. Whether SLC1A2 and/or other members of the SLC superfamily allow M. tuberculosis to access aspartate and multiply inside host cells remains to be evaluated. Another issue raised by our study is: how is nitrogen transferred from aspartate to other nitrogen-containing molecules in M. tuberculosis? Once acquired by the bacillus through its unique aspartate importer AnsP1, we showed this amino acid species mostly serves as a nitrogen provider and barely enters carbon metabolism through the Krebs cycle (Gouzy et al., 2013). Nitrogen assimilation from aspartate must rely on transamination steps allowing the transfer of aspartate-derived nitrogen to glutamate, which in turn, together with glutamine, provides nitrogen to most of biosynthesis pathways. In M. tuberculosis, two aspartate transaminases, called AspB and AspC, are predicted to mediate nitrogen transfer from aspartate to glutamate (Cole et al., 1998). Interestingly, the aspC gene is thought to be essential in M. tuberculosis (Sassetti et al., 2003), which may indicate that AspC is involved mostly in aspartate biosynthesis, rather than in aspartate catabolism and glutamate synthesis. Genetic inactivation of aspC may thus result in aspartate auxotrophy. The aspB gene, on the opposite, is not essential in vitro (Sassetti et al., 2003). Interestingly, aspB is expressed at higher level in bacteria withstanding conditions mimicking the intracellular environment (Fisher et al., 2002). AspB may thus be involved in aspartate-derived nitrogen assimilation during infection, and may be required for M. tuberculosis virulence. Noteworthy, AspB and AspC possess two homologue proteins, called Rv0858c and Rv1178, that are predicted to act as aspartate aminotransferases, and that might provide alternative pathways for aspartate-derived nitrogen assimilation in the TB bacillus (Cole et al., 1998). Finally, our study suggests aspartate is an important metabolite required for mycobacterial virulence, and this raises several questions regarding the therapeutic potential of inhibitors of enzymes using aspartate as a substrate. In addition to serve as a nitrogen provider, aspartate possesses non-redundant and essential functions in bacterial metabolism. As a consequence, several enzymes involved in aspartate metabolism, namely the aspartate decarboxylase PanD, the aspartokinase ASK, and the aspartate-β-semialdehyde dehydrogenase ASD, are considered promising targets for novel antituberculous compounds (Shafiani et al., 2005; Gopalan et al., 2006; Chaitanya et al., 2010; Sharma et al., 2012a,b). PanD is the sole enzyme in M. tuberculosis mediating the decarboxylation of aspartate, which allows the formation of vitamin B5, a precursor of the coenzyme A (CoA) co-factor. CoA is an acyl-carrier co-enzyme involved in fatty acid metabolism and is essential for the viability of several microorganisms, including M. tuberculosis (Ambady et al., 2012). Consequently, deletion of the panD gene was shown to result in a severe attenuation of M. tuberculosis virulence in the mouse model (Sambandamurthy et al., 2002). Another essential pathway relying on aspartate metabolism is the synthesis of amino acids belonging to the aspartate amino acid family, namely methionine, threonine and isoleucine, and involving the ASK and ASD enzymes, that play a unique and essential part in aspartate incorporation into these biosynthesis pathways. Furthermore, in addition to their role in the biosynthesis of these amino acids, both enzymes are involved in the synthesis of diaminopimelic acid, an intermediate of the lysine biosynthesis pathway, which is an essential constituent of the bacterial cell wall constituent peptidoglycan. As a consequence, ASK and ASD are attractive drug targets for new antituberculous molecules since i/ they possess no homologue in humans, and ii/ their inhibition in M. tuberculosis may cause both amino acid auxotrophy and cell wall destruction (Shafiani et al., 2005; Chaitanya et al., 2010). In conclusion, we believe aspartate acquisition and assimilation pathways should be further considered as promising targets for novel anti-TB therapies.

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.

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 bodys commensal bacteria during airway infectious diseases, including recent evidence highlighting their local (lung) or distal (gut) contribution in this process.