Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.     

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome–lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C‐terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non‐pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.     

ESX‐1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis

Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT‐6). This small protein, which is secreted by the type VII secretion system ESX‐1 (T7SS/ESX‐1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock‐out or knock‐in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX‐1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.     

Molecular analysis of the prokaryotic ubiquitin‐like protein (Pup) conjugation pathway in Mycobacterium tuberculosis

Proteins targeted for degradation by the Mycobacterium proteasome are post‐translationally tagged with prokaryotic ubiquitin‐like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed ‘pupylation’, Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild‐type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site‐directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.     

Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria

The ESX‐1 secretion system is required for pathogenicity of Mycobacterium tuberculosis (Mtb). Despite considerable research, little is known about the structural components of ESX‐1, or how these proteins are assembled into the active secretion apparatus. Here, we exploit the functionally related ESX‐1 apparatus of Mycobacterium smegmatis (Ms) to show that fluorescently tagged proteins required for ESX‐1 activity consistently localize to the cell pole, identified by time‐lapse fluoro‐microscopy as the non‐septal (old) pole. Deletions in Msesx1 prevented polar localization of tagged proteins, indicating the need for specific protein–protein interactions in polar trafficking. Remarkably, expression of the Mtbesx1 locus in Msesx1 mutants restored polar localization of tagged proteins, indicating establishment of the MtbESX‐1 apparatus in M. smegmatis. This observation illustrates the cross‐species conservation of protein interactions governing assembly of ESX‐1, as well as polar localization. Importantly, we describe novel non‐esx1‐encoded proteins, which affect ESX‐1 activity, which colocalize with ESX‐1, and which are required for ESX‐1 recruitment and assembly. This analysis provides new insights into the molecular assembly of this important determinant of Mtb virulence.     

Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence.     

Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.     

The Streptococcus pyogenes orphan protein tyrosine phosphatase,SP‐PTP,possesses dual specificity and essential virulence regulatory functions

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP‐PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP‐PTP is, therefore, questionable. Here, we demonstrate that SP‐PTP possesses dual phosphatase specificity for Tyr‐ and Ser/Thr‐phosphorylated GAS proteins, such as Ser/Thr kinase (SP‐STK) and the SP‐STK‐phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP‐PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP‐PTP displayed increased Ser‐/Thr‐/Tyr‐phosphorylation. SP‐PTP also differentially regulates the expression of ～50% of the total GAS genes, including several virulence genes potentially through the two‐component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP‐PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr‐phosphorylation in GAS and the relevance of SP‐PTP as an important therapeutic target.     

Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis

Strigolactones (SL) fulfil important roles in plant development and stress tolerance. Here, we characterized the role of SL in the dark chilling tolerance of pea and Arabidopsis by analysis of mutants that are defective in either SL synthesis or signalling. Pea mutants (rms3, rms4, and rms5) had significantly greater shoot branching with higher leaf chlorophyll a/b ratios and carotenoid contents than the wild type. Exposure to dark chilling significantly decreased shoot fresh weights but increased leaf numbers in all lines. Moreover, dark chilling treatments decreased biomass (dry weight) accumulation only in rms3 and rms5 shoots. Unlike the wild type plants, chilling‐induced inhibition of photosynthetic carbon assimilation was observed in the rms lines and also in the Arabidopsis max3‐9, max4‐1, and max2‐1 mutants that are defective in SL synthesis or signalling. When grown on agar plates, the max mutant rosettes accumulated less biomass than the wild type. The synthetic SL, GR24, decreased leaf area in the wild type, max3‐9, and max4‐1 mutants but not in max2‐1 in the absence of stress. In addition, a chilling‐induced decrease in leaf area was observed in all the lines in the presence of GR24. We conclude that SL plays an important role in the control of dark chilling tolerance.     

Structural basis for substrate recognition and inhibition of prephenate aminotransferase from Arabidopsis

Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (PAT) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition of PAT are currently unknown. Here we present the X‐ray crystal structures of the wild‐type and various mutants of PAT from Arabidopsis thaliana (AtPAT). Steady‐state kinetic and ligand‐binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution of PAT activity among class Ib AAT enzymes. Structures of AtPAT mutants co‐crystallized with either α‐ketoglutarate or pyridoxamine 5′‐phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor of PAT from A. thaliana and Antirrhinum majus plants as well as the bacterium Chlorobium tepidum, uncovering a potential new effector of PAT.     

A new role for glutathione in the regulation of root architecture linked to strigolactones

Reduced glutathione (GSH) is required for root development, but its functions are not characterized. The effects of GSH depletion on root development were therefore studied in relation to auxin and strigolactone (SL) signalling using a combination of molecular genetic approaches and pharmacological techniques. Lateral root (LR) density was significantly decreased in GSH synthesis mutants (cad2‐1, pad2‐, rax1‐), but not by the GSH synthesis inhibitor, buthionine sulfoximine (BSO). BSO‐induced GSH depletion therefore did not influence root architecture in the same way as genetic impairment. Root glutathione contents were similar in the wild‐type seedlings and max3‐9 and max4‐1 mutants that are deficient in SL synthesis and in the SL‐signalling mutant, max2‐1. BSO‐dependent inhibition of GSH synthesis depleted the tissue GSH pool to a similar extent in the wild‐type and SL synthesis mutants, with no effect on LR density. The application of the SL analogue GR24 increased root glutathione in the wild‐type, max3‐9 and max4‐1 seedlings, but this increase was absent from max2‐1. Taken together, these data establish a link between SLs and the GSH pool that occurs in a MAX2‐dependent manner.     

Regulation of the KstR2 regulon of Mycobacterium tuberculosis by a cholesterol catabolite

Cholesterol catabolism is widespread in actinobacteria and is critical for Mycobacterium tuberculosis (Mtb) virulence. Catabolism of steroid nucleus rings C and D is poorly understood: it is initiated by the CoA thioesterification of 3aα‐H‐4α(3′‐propanoate)‐7aβ‐methylhexahydro‐1,5‐indanedione (HIP) by FadD3, whose gene is part of the KstR2 regulon. In Mtb, genes of this regulon were upregulated up to 30‐ and 22‐fold during growth on cholesterol and HIP, respectively, versus another minimal medium. In contrast, genes involved in degrading the cholesterol side‐chain and nucleus rings A and B were only upregulated during growth on cholesterol. Similar results were obtained in Rhodococcus jostii RHA1. Moreover, the regulon was not upregulated in a ΔfadD3 mutant unable to produce HIP‐CoA. In electrophoretic mobility shift assays, HIP‐CoA relieved the binding of KstR2_Mtb to each of three KstR2 boxes: CoASH, HIP and a related CoA thioester did not. Inspection of the structure of KstR2_RHA1 revealed no obvious HIP‐CoA binding pocket. The results establish that Mtb can catabolize the entire cholesterol molecule and that HIP‐CoA is an effector of KstR2. They further indicate that KstR2 specifically represses the expression of the HIP degradation genes in actinobacteria, which encode a lower pathway involved in the catabolism of multiple steroids.     

A novel F420‐dependent anti‐oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F₄₂₀‐dependent anti‐oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F₄₂₀‐deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD⁺ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F₄₂₀^? mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin‐dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F₄₂₀H₂‐dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F₄₂₀H₂‐specific obligate two‐electron reduction of endogenous quinones, thereby competing with the one‐electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F₄₂₀ biosynthesis pathway or Fqr‐class proteins as a mechanism to potentiate the action of bactericidal agents.     

Impact of bedaquiline and capreomycin on the gene expression patterns of multidrug-resistant Mycobacterium tuberculosis H37Rv strain and understanding the molecular mechanism of antibiotic resistance

The emergence of multidrug resistance (MDR), extensively drug-resistant, and total drug-resistant Mycobacterium tuberculosis (Mtb) strains have hampered the treatment of tuberculosis (TB). Capreomycin and Bedaquiline are currently used for MDR-TB treatment. To understand the impact of these antibiotics on Mtb genes, we have curated the gene expression data where the Mtb cultures were exposed to the Bedaquiline and Capreomycin. Based on the P value cut off (<0.05) and logFC (<−0.5 and >+0.5) values, we have selected the top differentially expressed genes during the antibiotic exposures. We have observed that the top differentially expressed Mtb genes were related to universal stress genes, two-component regulatory systems, and drug efflux pumps. We have curated the Mtb gene datasets and carried out the functional over-representation analysis using the individual gene expression values. We further, constructed the gene interaction networks of antibiotic resistance genes and virulence genes of Mtb to understand the impact of the antibiotics at the molecular level and thus to understand the antimicrobial resistance and virulence patterns. Our study elucidates the impact of antibiotics on the Mtb genes at the molecular level and the positively enriched pathways, operons, and regulons data are helpful in understanding the resistance patterns in Mtb. The upregulated genes during the exposure of Bedaquiline and Capreomycin can be considered as potent drug targets for the development of new anti-TB drugs.     

Mycobacterium tuberculosis protein ESAT‐6 is a potent activator of the NLRP3/ASC inflammasome

Interleukin‐1β (IL‐1β) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL‐1β secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase‐1 activity and promotes the secretion of IL‐1β. This stimulation requires live intracellular bacteria expressing a functional ESX‐1 secretion system. ESAT‐6, an ESX‐1 substrate implicated in membrane damage, is both necessary and sufficient for caspase‐1 activation and IL‐1β secretion. ESAT‐6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase‐1 activation largely by perturbing host cell membranes. Using a high‐throughput shRNA‐based screen we found that numerous NOD‐like receptors (NLRs) and CARD domain‐containing proteins (CARDs) were important for IL‐1β secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase‐1 form an infection‐inducible inflammasome complex that is essential for IL‐1β secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT‐6, and the subsequent IL‐1β response is regulated by a number of NLR/CARD proteins.