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The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled. [ABSTRACT FROM AUTHOR]

Summary Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis , the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA , plcB , plcC and plcD . However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild-type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis . Functional complementation of the Δ plcABC triple mutant with the individual plcA , plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis . RT-PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus. [ABSTRACT FROM AUTHOR]

Summary Mycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin-binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope. Four different fbp genes are found in the genome of M. tuberculosis , but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown. We have shown previously that, in the heterologous host, Corynebacterium glutamicum , FbpA, FbpB and FbpC can all add mycoloyl residues to the cell wall arabinogalactan and that, in M. tuberculosis , the cell wall mycoloylation decreases by 40% when fbpC is knocked out. To investigate whether the remaining 60% mycoloylation came from the activity of FbpA and/or FbpB, fbpA - and fbpB -inactivated mutant strains were biochemically characterized and compared with the previously studied fbpC -disrupted mutant. Unexpectedly, both mutants produced normally mycoloylated cell walls. Overproduction of FbpA, FbpB or FbpC, but not FbpD, in the fbpC -inactivated mutant strain of M. tuberculosis restored both the cell wall-linked mycolate defect and the outer cell envelope permeability barrier property. These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps. In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC -inactivated mutant. Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan... [ABSTRACT FROM AUTHOR]

Describes the characteristics of an insertion sequence isolated from Mycobacterium smegmatis. DNA isolation and manipulation; Southern blots and hybridizations; DNA sequencing; Sequence computer analysis.

Studies the mycobacterial insertional mutant libraries constructed by transposition. Construction of plasmid CG79; Transposition mediated by pCG79; Applications and uses of the mutant libraries.

Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, and R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth and reduced virulence. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth, at least in part through the quinone oxidoreductase NQO1, and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose boosting of the NRF2-NQO1 axis as a potential host-directed strategy to improve Mabs infection control. Author summary: Pulmonary infection by Non Tuberculosis Mycobacteria is a rising concern for patients with cystic fibrosis (CF), especially Mycobacterium abscessus (Mabs). Mabs exists as two morphotypes. CF patients are generally infected by the S morphotype present in the environment, which can switch to the R morphotype displaying higher virulence. Due to its resistance to antibiotics, treatments againt Mabs often fail, calling for complementary therapeutical strategies. Here we adapted the human airway organoid technology to model Mabs infection in the context of CF, decipher mechanisms of host-pathogen interaction that can be pharmacologically targeted to improve infection control. We found that Mabs R induces higher host oxidative stress and cell death, hallmarks of its virulence, which are enhanced in the CF context. Boosting the host oxidative pathway using antioxidants improves infection control by a frontline antibiotic. Our study provides CF patient-derived airway organoids as a relevant human-based, animal-free system for CF-driven Mabs infection and evaluation of innovative therapeutic strategies. [ABSTRACT FROM AUTHOR]

The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii-like ancestor, remain poorly investigated. In MTB, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M. canettii relative to MTB, impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M. canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M. canettii strain, displays lower expression of PhoP-induced genes than MTB. Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M. canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M. canettii strains than in M. tuberculosis. Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host. Author summary: Mycobacterium tuberculosis (MTB) likely emerged from an environmental ancestor by adaptation of endogenous pathways enhancing its capacity to colonize and transmit within the human host. The closest extant relatives of the MTB ancestor are the Mycobacterium canettii strains which are opportunist pathogens responsible for rare human tuberculosis cases in East-Africa. Here we found that the sensor protein of the important two-component regulatory system PhoPR displays amino acid substitutions that modulate its activity in M. canettii in comparison to MTB. These mutations impact the production and secretion of virulence factors important for multiplication within the host and cough induction. Therefore, our findings support the hypothesis that variants of the PhoPR regulatory system were selected as key evolutionary adaptations for enhancing the epidemic capacity of the tuberculosis bacilli. [ABSTRACT FROM AUTHOR]

Background: Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure‐function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. Results: We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. Conclusions: The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself. [ABSTRACT FROM AUTHOR]

Respiratory infections remain a major global health concern. Tuberculosis is one of the top 10 causes of death worldwide, while infections with Non‐Tuberculous Mycobacteria are rising globally. Recent advances in human tissue modeling offer a unique opportunity to grow different human "organs" in vitro, including the human airway, that faithfully recapitulates lung architecture and function. Here, we have explored the potential of human airway organoids (AOs) as a novel system in which to assess the very early steps of mycobacterial infection. We reveal that Mycobacterium tuberculosis (Mtb) and Mycobacterium abscessus (Mabs) mainly reside as extracellular bacteria and infect epithelial cells with very low efficiency. While the AO microenvironment was able to control, but not eliminate Mtb, Mabs thrives. We demonstrate that AOs responded to infection by modulating cytokine, antimicrobial peptide, and mucin gene expression. Given the importance of myeloid cells in mycobacterial infection, we co‐cultured infected AOs with human monocyte‐derived macrophages and found that these cells interact with the organoid epithelium. We conclude that adult stem cell (ASC)‐derived AOs can be used to decipher very early events of mycobacteria infection in human settings thus offering new avenues for fundamental and therapeutic research. [ABSTRACT FROM AUTHOR]

Successful control of Mycobacterium tuberculosis (Mtb) infection by macrophages relies on immunometabolic reprogramming, where the role of fatty acids (FAs) remains poorly understood. Recent studies unraveled Mtb's capacity to acquire saturated and monounsaturated FAs via the Mce1 importer. However, upon activation, macrophages produce polyunsaturated fatty acids (PUFAs), mammal-specific FAs mediating the generation of immunomodulatory eicosanoids. Here, we asked how Mtb modulates de novo synthesis of PUFAs in primary mouse macrophages and whether this benefits host or pathogen. Quantitative lipidomics revealed that Mtb infection selectively activates the biosynthesis of ?6 PUFAs upstream of the eicosanoid precursor arachidonic acid (AA) via transcriptional activation of Fads2. Inhibiting FADS2 in infected macrophages impaired their inflammatory and antimicrobial responses but had no effect on Mtb growth in host cells nor mice. Using a click-chemistry approach, we found that Mtb efficiently imports ?6 PUFAs via Mce1 in axenic culture, including AA. Further, Mtb preferentially internalized AA over all other FAs within infected macrophages by mechanisms partially depending on Mce1 and supporting intracellular persistence. Notably, IFN? repressed de novo synthesis of AA by infected mouse macrophages and restricted AA import by intracellular Mtb. Together, these findings identify AA as a major FA substrate for intracellular Mtb, whose mobilization by innate immune responses is opportunistically hijacked by the pathogen and downregulated by IFN?. [ABSTRACT FROM AUTHOR]

Gene disruption experiments play an important role in the functional characterization of genes in mycobacteria and rely mostly on the use of one or two antibiotic resistance markers. We have developed a system for mycobacteria which features both the advantages of the use of antibiotic resistance markers for gene disruption experiments and the ability to efficiently rescue the marker leaving an unmarked mutation on the chromosome. This new genetic tool relies on the transposon γδ site-specific recombination system. A res-ΩKm-res cassette was used to generate an insertional mutation by allelic exchange both in Mycobacterium smegmatis and Mycobacterium bovis BCG. Upon expression in the mutated strains of tnpR, the transposon γδ resolvase gene, res-ΩKm-res, was excised efficiently leaving behind a single res sequence at the mutated locus. A plasmid was engineered allowing expression of tnpR from an easily curable mycobacterial vector. This system will be useful for simple construction of unmarked mutations or repeated use of the same antibiotic marker to generate multiple mutants. [Copyright &y& Elsevier]

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity. Author summary: The epidemic efficiency of tuberculosis bacilli is determined by their capacity to transmit via aerosol. Currently, the bacterial functions that favor Mycobacterium tuberculosis seeding in the lung of naïve host remain mostly unknown. Here we implemented a genome-wide approach to identify M. tuberculosis mutants deficient for seeding and early replication in the lung of mice. In addition to genes known to encode virulence factors, we identified three genes not previously described. We used complementary approaches to characterize the phenotype of a M. tuberculosis mutant with insertion within the rv0180c gene. We found that this mutant is impaired for seeding in the lung of mice and for invasion and replication in human macrophages. In macrophages, the defect relies on a lack of engagement of CR3 receptor. Although we did not detect any difference between the wild type strain and the rv0180c mutant with regard to potential CR3-ligand, we found that the bacterial cell envelope is altered in the rv0180c mutant. Our study provides new insight into bacterial genes required for early interaction of M. tuberculosis with the host and perspective to understand the bacterial functions enhancing infectivity. [ABSTRACT FROM AUTHOR]

Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors. [ABSTRACT FROM AUTHOR]

One central question surrounding the biosynthesis of fatty acids and polyketide‐derived natural products is how the 4′‐phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4′‐phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post‐translational modification of modular megasynthases. [ABSTRACT FROM AUTHOR]

Trehalose polyphleates (TPP) are high-molecular-weight, surface-exposed glycolipids present in a broad range of nontuberculous mycobacteria. These compounds consist of a trehalose core bearing polyunsaturated fatty acyl substituents (called phleic acids) and a straight-chain fatty acid residue and share a common basic structure with trehalose-based glycolipids produced by Mycobacterium tuberculosis. TPP production starts in the cytosol with the formation of a diacyltrehalose intermediate. An acyltransferase, called PE, subsequently catalyzes the transfer of phleic acids onto diacyltrehalose to form TPP, and an MmpL transporter promotes the export of TPP or its precursor across the plasma membrane. PE is predicted to be an anchored membrane protein, but its topological organization is unknown, raising questions about the subcellular localization of the final stage of TPP biosynthesis and the chemical nature of the substrates that are translocated by the MmpL transporter. Here, using genetic, biochemical, and proteomic approaches, we established that PE of Mycobacterium smegmatis is exported to the cell envelope following cleavage of its signal peptide and that this process is required for TPP biosynthesis, indicating that the last step of TPP formation occurs in the outer layers of the mycobacterial cell envelope. These results provide detailed insights into the molecular mechanisms controlling TPP formation and transport to the cell surface, enabling us to propose an updated model of the TPP biosynthetic pathway. Because the molecular mechanisms of glycolipid production are conserved among mycobacteria, these findings obtained with PE from M. smegmatis may offer clues to glycolipid formation in M. tuberculosis. [ABSTRACT FROM AUTHOR]

Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce 4 operon of MAP. Finally, our results showed that the deletion of mce 4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modeling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multiscale molecular modeling and 31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregates in the stalks formed between 2 opposing lipid bilayers. Infection of macrophages pretreated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological response of macrophages. [ABSTRACT FROM AUTHOR]

Mycobacterium leprae, an obligate intracellular bacillus, infects Schwann cells (SCs), leading to peripheral nerve damage, the most severe leprosy symptom. In the present study, we revisited the involvement of phenolic glycolipid I (PGL I), an abundant, private, surface M. leprae molecule, in M. leprae-SC interaction by using a recombinant strain of M. bovis BCG engineered to express this glycolipid. We demonstrate that PGL I is essential for bacterial adhesion and SC internalization. We also show that live mycobacterium-producing PGL I induces the expression of the endocytic mannose receptor (MR/CD206) in infected cells in a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent manner. Of note, blocking mannose recognition decreased bacterial entry and survival, pointing to a role for this alternative recognition pathway in bacterial pathogenesis in the nerve. Moreover, an active crosstalk between CD206 and the nuclear receptor PPARγ was detected that led to the induction of lipid droplets (LDs) formation and prostaglandin E2 (PGE2), previously described as fundamental players in bacterial pathogenesis. Finally, this pathway was shown to induce IL-8 secretion. Altogether, our study provides evidence that the entry of live M. leprae through PGL I recognition modulates the SC phenotype, favoring intracellular bacterial persistence with the concomitant secretion of inflammatory mediators that may ultimately be involved in neuroinflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary Mycobacteria synthesize a variety of structurally related glycolipids with major biological functions. Common themes have emerged for the biosynthesis of these glycolipids, including several families of proteins. Genes encoding these proteins are usually clustered on bacterial chromosomal islets dedicated to the synthesis of one glycolipid family. Here, we investigated the function of a cluster of five genes widely distributed across non-tuberculous mycobacteria. Using defined mutant analysis and in-depth structural characterization of glycolipids from wild-type or mutant strains of Mycobacterium smegmatis and Mycobacterium abscessus , we established that they are involved in the formation of trehalose polyphleates (TPP), a family of compounds originally described in Mycobacterium phlei . Comparative genomics and lipid analysis of strains distributed along the mycobacterial phylogenetic tree revealed that TPP is synthesized by a large number of non-tuberculous mycobacteria. This work unravels a novel glycolipid biosynthetic pathway in mycobacteria and extends the spectrum of bacteria that produce TPP. [ABSTRACT FROM AUTHOR]

Summary Mycolate-containing compounds constitute major strategic elements of the protective coat surrounding the tubercle bacillus. We have previously shown that FAAL32-Pks13 polyketide synthase catalyzes the condensation reaction, which produces α-alkyl β-ketoacids, direct precursors of mycolic acids. In contrast to the current biosynthesis model, we show here that Pks13 catalyzes itself the release of the neosynthesized products and demonstrate that this function is carried by its thioesterase-like domain. Most importantly, in agreement with the prediction of a trehalose-binding pocket in its catalytic site, this domain exhibits an acyltransferase activity and transfers Pks13’s products onto an acceptor molecule, mainly trehalose, leading to the formation of the trehalose monomycolate precursor. Thus, this work allows elucidation of the hinge step of the mycolate-containing compound biosynthesis pathway. Above all, it highlights a unique mechanism of transfer of polyketide synthase products in mycobacteria, which is distinct from the conventional intervention of the discrete polyketide-associated protein (Pap)-type acyltransferases. [ABSTRACT FROM AUTHOR]

Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6770 element upstream of the phoPR locus may completely revert the phoPR-bovis-assocS-ated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB. [ABSTRACT FROM AUTHOR]

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. [ABSTRACT FROM AUTHOR]

Several specific lipids of the cell envelope are implicated in the pathogenesis of M. tuberculosis ( Mtb), including phthiocerol dimycocerosates ( DIM) that have clearly been identified as virulence factors. Others, such as trehalose-derived lipids, sulfolipids ( SL), diacyltrehaloses ( DAT) and polyacyltrehaloses ( PAT), are believed to be essential for Mtb virulence, but the details of their role remain unclear. We therefore investigated the respective contribution of DIM, DAT/ PAT and SL to tuberculosis by studying a collection of mutants, each with impaired production of one or several lipids. We confirmed that among those with a single lipid deficiency, only strains lacking DIM were affected in their replication in lungs and spleen of mice in comparison to the WT Mtb strain. We found also that the additional loss of DAT/ PAT, and to a lesser extent of SL, increased the attenuated phenotype of the DIM-less mutant. Importantly, the loss of DAT/ PAT and SL in a DIM-less background also affected Mtb growth in human monocyte-derived macrophages ( hMDMs). Fluorescence microscopy revealed that mutants lacking DIM or DAT/ PAT were localized in an acid compartment and that bafilomycin A1, an inhibitor of phagosome acidification, rescued the growth defect of these mutants. These findings provide evidence for DIM being dominant virulence factors that mask the functions of lipids of other families, notably DAT/ PAT and to a lesser extent of SL, which we showed for the first time to contribute to Mtb virulence. [ABSTRACT FROM AUTHOR]

Highlights: [•] Construction, characterization and preclinical studies of MTBVAC, an attenuated Mycobacterium tuberculosis. [•] MTBVAC vaccine candidate is genetically engineered to fulfil the Geneva consensus (Vaccine Kamath et al., 2005). [•] MTBVAC is the first live attenuated M. tuberculosis vaccine to enter clinical evaluation. [•] MTBVAC Clinical trials started in January 2013 (Swissmedic reference MTBVAC 2012GT1002). [ABSTRACT FROM AUTHOR]

Abstract: The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1–10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4′-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg2+, essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042–2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain. [Copyright &y& Elsevier]

A posttranslational protein O-mannosylation process resembling that found in fungi and animals has been reported in the major human pathogen Mycobacterium tuberculosis (Mtb) and related actinobacterja. However, the role and incidence of this process, which is essential in eukaryotes, have never been explored in Mtb. We thus analyzed the impact of interrupting O-mannosylation in the nonpathogenic saprophyte Mycobacterium smegmatis and in the human pathogen Mtb by inactivating the respective putative protein mannosyl transferase genes Msmeg_5447 and RvlOO2c. Loss of protein O-mannosylation in both mutant strains was unambiguously demonstrated by efficient mass spectrometry-based glycoproteomics analysis. Unexpectedly, although the M. smegmatis phenotype was unaffected by the lack of man no-proteins, the Mtb mutant had severely impacted growth in vitro and in cellulo associated with a strong attenuation of its pathogenicity in immunocompromised mice. These data are unique in providing evidence of the biological significance of protein O-mannosylation in mycobacteria and demonstrate the crucial contribution of this protein posttranslational modification to Mtb virulence in the host. [ABSTRACT FROM AUTHOR]

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3. [ABSTRACT FROM AUTHOR]

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4′-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections. [ABSTRACT FROM AUTHOR]

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis. [ABSTRACT FROM AUTHOR]

The six major genetic lineages of Mycobacterium tuberculosis are strongly associated with specific geographical regions, but their relevance to bacterial virulence and the clinical consequences of infection are unclear. Previously, we found that in Vietnam, East Asian/Beijing and Indo-Oceanic strains were significantly more likely to cause disseminated tuberculosis with meningitis than those from the Euro-American lineage. To investigate this observation we characterised 7 East Asian/Beijing, 5 Indo-Oceanic and 6 Euro-American Vietnamese strains in bone-marrow-derived macrophages, dendritic cells and mice. East Asian/Beijing and Indo-Oceanic strains induced significantly more TNF-&agr; and IL-1&bgr; from macrophages than the Euro-American strains, and East Asian/Beijing strains were detectable earlier in the blood of infected mice and grew faster in the lungs. We hypothesised that these differences were induced by lineage-specific variation in cell envelope lipids. Whole lipid extracts from East Asian/Beijing and Indo-Oceanic strains induced higher concentrations of TNF-&agr; from macrophages than Euro-American lipids. The lipid extracts were fractionated and compared by thin layer chromatography to reveal a distinct pattern of lineageassociated profiles. A phthiotriol dimycocerosate was exclusively produced by East Asian/Beijing strains, but not the phenolic glycolipid previously associated with the hyper-virulent phenotype of some isolates of this lineage. All Indo-Oceanic strains produced a unique unidentified lipid, shown to be a phenolphthiocerol dimycocerosate dependent upon an intact pks15/1 for its production. This was described by Goren as the 'attenuation indictor lipid' more than 40 years ago, due to its association with less virulent strains from southern India. Mutation of pks15/1 in a representative Indo-Oceanic strain prevented phenolphthiocerol dimycocerosate synthesis, but did not alter macrophage cytokine induction. Our findings suggest that the early interactions between M. tuberculosis and host are determined by the lineage of the infecting strain; but we were unable to show these differences are driven by lineage-specific cell-surface expressed lipids. [ABSTRACT FROM AUTHOR]

Phthiocerol and phthiodiolone dimycocerosates (DIMs) and phenolic glycolipids (PGLs) are complex lipids located at the cell surface of Mycobacterium tuberculosis that play a key role in the pathogenicity of tuberculosis. Most of the genes involved in the biosynthesis of these compounds are clustered on a region of the M. tuberculosis chromosome, the so-called DIM + PGL locus. Among these genes, four ORFs encode FadD proteins, which activate and transfer biosynthetic intermediates onto various polyketide synthases that catalyze the formation of these lipids. In this study, we investigated the roles of FadD22, FadD26 and FadD29 in the biosynthesis of DIMs and related compounds. Biochemical characterization of the lipids produced by a spontaneous Mycobacterium bovis BCG mutant harboring a large deletion within fadD26 revealed that FadD26 is required for the production of DIMs but not of PGLs. Additionally, using allelic exchange recombination, we generated an unmarked M. tuberculosis mutant containing a deletion within fadD29. Biochemical analyses of this strain revealed that, like fadD22, this gene encodes a protein that is specifically involved in the biosynthesis of PGLs, indicating that both FadD22 and FadD29 are responsible for one particular reaction in the PGL biosynthetic pathway. These findings were also supported by in vitro enzymatic studies showing that these enzymes have different properties, FadD22 displaying a p-hydroxybenzoyl-AMP ligase activity, and FadD29 a fatty acyl-AMP ligase activity. Altogether, these data allowed us to precisely define the functions fulfilled by the various FadD proteins encoded by the DIM + PGL cluster. [ABSTRACT FROM AUTHOR]

Corynebacterineae are gram-positive bacteria that possess a true outer membrane composed of mycolic acids and other lipids. Little is known concerning the modulation of mycolic acid composition and content in response to changes in the bacterial environment, especially temperature variations. To address this question, we investigated the function of the Rv3802c gene, a gene conserved in Corynebacterineae and located within a gene cluster involved in mycolic acid biosynthesis. We showed that the Rv3802 ortholog is essential in Mycobacterium smegmatis, while its Corynebacterium glutamicum ortholog, NCgl2775, is not. We provided evidence that the NCgl2775 gene is transcriptionally induced under heat stress conditions, and while the corresponding protein has no detectable activity under normal growth conditions, the increase in its expression triggers an increase in mycolic acid biosynthesis concomitant with a decrease in phospholipid content. We demonstrated that these lipid modifications are part of a larger outer membrane remodeling that occurs in response to exposure to a moderately elevated temperature (42°C). In addition to showing an increase in the ratio of saturated corynomycolates to unsaturated corynomycolates, our results strongly suggested that the balance between mycolic acids and phospholipids is modified inside the outer membrane following a heat challenge. Furthermore, we showed that these lipid modifications help the bacteria to protect against heat damage. The NCgl2775 protein and its orthologs thus appear to be a protein family that plays a role in the regulation of the outer membrane lipid composition of Corynebacterineae under stress conditions. We therefore propose to name this protein family the envelope lipids regulation factor (ElrF) family. [ABSTRACT FROM AUTHOR]

Lipooligosaccharides (LOS) are highly antigenic glycolipids produced by a number of Mycobacterium species, which include "M. canettii," a member of the M. tuberculosis complex, and the opportunistic pathogens M. marinum and M. kansasii. The various LOS share a core composed of trehalose esterified by at least 1 mole of polymethyl-branched fatty acid (PMB-FA) and differ from one another by their oligosaccharide extensions. In this study, we identified a cluster of genes, MSMEG_4727 through MSMEG_4741, likely involved in the synthesis of LOS in M. smegmatis. Disruption of MSMEG_4727 (the ortholog of pks5 of M. tuberculosis), which encodes a putative polyketide synthase, resulted in the concomitant abrogation of the production of both PMB-FA and LOS in the mutant strain. Complementation of the mutant with the wild-type gene fully restored the phenotype. We also showed that, in contrast to the case for "M. canettii" and M. marinum, LOS are located in deeper compartments of the cell envelope of M. smegmatis. The availability of two mycobacterial strains differing only in LOS production should help in defining the biological role(s) of this important glycolipid. [ABSTRACT FROM AUTHOR]

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 cholesteroldepleting 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. [ABSTRACT FROM AUTHOR]

Phenolic glycolipids (PGL) play a major role in the virulence of mycobacteria, notably in strains of the Mycobacterium tuberculosis complex and in Mycobacterium leprae. The structure of the carbohydrate domain of these compounds is highly variable, and the genetic bases for these variations remain unknown. We demonstrated that the monoglycosylated PGL formed by Mycobacterium bovis differs from the triglycosylated PGL synthesized by M. tuberculosis (PGL-tb) because of the following two genetic defects: a frameshift mutation within the gene Rv2958c, encoding a glycosyltransferase involved in the transfer of the second rhamnbsyl residue of the PGL-tb, and a deletion of a region that encompasses two genes, which encode a GDP-D-mannose 4,6-dehydratase and a GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/reductase, required for the formation of activated L-fucose. Expression of these three genes in M. bovis BCG allowed synthesis of PGL-tb in this recombinant strain. Additionally, we showed that all M. bovis, Mycobacterium microti, Mycobacterium pinnipedii, and some Mycobacterium africanum strains harbor the same frameshift mutation in their Rv2958c orthologs. Consistently, the structure of PGLs purified from M. africanum (harboring the Rv2958c mutation) and M. pinnipedii strains revealed that these compounds are monoglycosylated PGL. These findings explain the specificity of PGL-tb production by some strains of the M. tuberculosis complex and have important implications for our understanding of the evolution of this complex. [ABSTRACT FROM AUTHOR]

Phthiocerol dimycocerosates and related compounds are important molecules in the biology of Mycobacterium tuberculosis, playing a key role in the permeability barrier and in pathogenicity. Both phthiocerol dimycocerosates, the major compounds, and phthiodiolone dimycocerosates, the minor constituents, are found in the cell envelope of M. tuberculosis, but their specific roles in the biology of the tubercle bacillus have not been established yet. According to the current model of their biosynthesis, phthiocerol is produced from phthiodiolone through a two-step process in which the keto group is first reduced and then methylated. We have previously identified the methyltransferase enzyme that is involved in this process, encoded by the gene Rv2952 in M. tuberculosis. In this study, we report the construction and biochemical analyses of an M. tuberculosis strain mutated in gene Rv2951c. This mutation prevents the formation of phthiocerol and phenolphthiocerol derivatives, but leads to the accumulation of phthiodiolone dimycocerosates and glycosylated phenolphthiodiolone dimycocerosates. These results provide the formal evidence that Rv2951c encodes the ketoreductase catalyzing the reduction of phthiodiolone and phenolphthiodiolone to yield phthiotriol and phenolphthiotriol, which are the substrates of the methyltransferase encoded by gene Rv2952. We also compared the resistance to SDS and replication in mice of the Rv2951c mutant, deficient in synthesis of phthiocerol dimycocerosates but producing phthiodiolone dimycocerosates, with those of a wild-type strain and a mutant without phthiocerol and phthiodiolone dimycocerosates. The results established the functional redundancy between phthiocerol and phthiodiolone dimycocerosates in both the protection of the mycobacterial cell and the pathogenicity of M. tuberculosis in mice. [ABSTRACT FROM AUTHOR]

Abstract: The (R)-specific 3-hydroxyacyl dehydratases/trans-enoyl hydratases are key proteins in the biosynthesis of fatty acids. In mycobacteria, such enzymes remain unknown, although they are involved in the biosynthesis of major and essential lipids like mycolic acids. First bioinformatic analyses allowed to identify a single candidate protein, namely Rv3389c, that belongs to the hydratases 2 family and is most likely made of a distinctive asymmetric double hot dog fold. The purified recombinant Rv3389c protein was shown to efficiently catalyze the hydration of (C8–C16) enoyl-CoA substrates. Furthermore, it catalyzed the dehydration of a 3-hydroxyacyl-CoA in coupled reactions with both reductases (MabA and InhA) of the acyl carrier protein (ACP)-dependent M. tuberculosis fatty acid synthase type II involved in mycolic acid biosynthesis. Yet, the facts that Rv3389c activity decreased in the presence of ACP, versus CoA, derivative and that Rv3389c knockout mutant had no visible variation of its fatty acid content suggested the occurrence of additional hydratase/dehydratase candidates. Accordingly, further and detailed bioinformatic analyses led to the identification of other members of the hydratases 2 family in M. tuberculosis. [Copyright &y& Elsevier]

Mycobacterium tuberculosis contains >20 enzymes that require activation by transfer of the 4′-phosphopantetheine moiety of CoA onto a conserved serine residue, a posttranslational modification catalyzed by 4′-phosphopantetheinyl transferases (PPTases). The modified proteins are involved in key metabolic processes such as cell envelope biogenesis and the production of virulence factors. We show that two PPTases conserved in all Mycobacterium spp. and in related genera activate two different subsets of proteins and are not functionally redundant. One enzyme, AcpS, activates the two fatty acid synthase systems of mycobacteria, whereas the other PPTase, PptT, acts on type-I polyketide synthases and non-ribosomal peptide synthases, both of which are involved in the biosynthesis of virulence factors. We demonstrate that both PPTases are essential for Mycobacterium smegmatis viability and that PptT is required for the survival of Mycobacterium bovis bacillus Calmette-Guérin. These enzymes are thus central to the biology of mycobacteria and for mycobacterial pathogenesis and represent promising targets for new antituberculosis drugs. [ABSTRACT FROM AUTHOR]

A few mycobacterial species, most of which are pathogenic for humans, produce dimycocerosates of phthiocerol (DIM) and of glycosylated phenolphthiocerol, also called phenolglycolipid (PGL), two groups of molecules shown to be important virulence factors. The biosynthesis of these molecules is a very complex pathway that involves more than 15 enzymatic steps and has just begun to be elucidated. Most of the genes known to be involved in these pathways are clustered on the chromosome of M. tuberculosis. Based on their amino acid sequences, we hypothesized that the proteins encoded by Rv2952 and Rv2959c, two open reading frames of this locus, are involved in the transfer of methyl groups onto various hydroxyl functions during the biosynthesis of DIM, PGL, and related p-hydroxybenzoic acid derivatives (p-HBAD). Using allelic exchange and site-specific recombination, we produced three recombinant strains of Mycobacterium tuberculosis carrying insertions in Rv2952 or Rv2959c. Analysis of these mutants revealed that (i) the protein encoded by Rv2952 is a methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL, respectively, (ii) Rv2959c is part of an operon including the newly characterized Rv2958c gene that encodes a glycosyltransferase also involved in PGL and p-HBAD biosynthesis, and (iii) the enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD produced by M. tuberculosis. These data further extend our understanding of the biosynthesis of important mycobacterial virulence factors and provide additional tools to decipher the molecular mechanisms of action of these molecules during the pathogenesis of tuberculosis. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans, produce highly specific long chain β-diols, the dimycocerosates of phthiocerol, and structurally related phenolic glycolipid (PGL) antigens, which are important virulence factors. In addition, M. tuberculosis also secretes glycosylated p-hydroxybenzoic acid methyl esters (p-HBAD) that contain the same carbohydrate moiety as the species-specific PGL of M. tuberculosis (PGL-tb). The genes involved in the biosynthesis of these compounds in M. tuberculosis are grouped on a 70-kilobase chromosomal fragment containing three genes encoding putative glycosyltransferases: Rv2957, Rv2958c, and Rv2962c. To determine the functions of these genes, three recombinant M. tuberculosis strains, in which these genes were individually inactivated, were constructed and biochemically characterized. Our results demonstrated that (i) the biosynthesis of PGL-tb and p-HBAD involves common enzymatic steps, (ii) the Rv2957, Rv2958c, and Rv2962c genes are involved in the formation of the glycosyl moiety of the two classes of molecules, and (iii) the product of Rv2962c catalyzes the transfer of a rhamnosyl residue onto p-hydroxybenzoic acid ethyl ester or phenolphthiocerol dimycocerosates, whereas the products of Rv2958c and Rv2957 add a second rhamnosyl unit and a fucosyl residue to form the species-specific triglycosyl appendage of PGL-tb and p-HBAD. The recombinant strains produced provide the tools to study the role of the carbohydrate domain of PGL-tb and p-HBAD in M. tuberculosis pathogenesis. [ABSTRACT FROM AUTHOR]

Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase , the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae. [ABSTRACT FROM AUTHOR]

Two-component regulatory proteins function in bacteria as sensory and adaptive factors in response to a wide range of environmental stimuli. Some two-component systems, such as PhoP/PhoQ, control transcription of key virulence genes essential for survival in host cells in diverse intracellular bacterial pathogens, including Salmonella sp., Shigella sp. and Yersinia sp. In this study, we have disrupted the phoP gene from Mycobacterium tuberculosis, which codes for a putative transcription regulator factor of the two-component system PhoP/PhoR. The phoP mutant strain exhibited impaired multiplication when cultured in mouse bone marrow-derived macrophages. However, the mutation did not appear to affect survival of the organisms adversely inside macrophages. The mutant strain was also attenuated in vivo in a mouse infection model, with impaired growth observed in the lungs, livers and spleens. The results suggest that the phoP gene is required for intracellular growth of M. tuberculosis but is not essential for persistence of the bacilli. [ABSTRACT FROM AUTHOR]

Tuberculosis remains the greatest cause of death worldwide due to a single pathogen. In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis , a functional genomic approach was developed. A library of signature-tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice. From 1927 mutants tested, 16 were attenuated for their virulence. The insertions harboured by the selected mutants were mapped on the M. tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane. Four independent mutations identified a cluster of virulence genes located on a 50 kb chromosomal region. These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens. The interaction of five mutant strains with mouse bone marrow macrophages was investigated. These five mutants were still able to multiply in this cell type. However, in three cases, there was a growth defect in comparison with the wild-type strain. The other two strains exhibited no clear difference from the virulent strain, MT103, in this model. This study, which is the first global research of virulence factors of M. tuberculosis , opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host. [ABSTRACT FROM AUTHOR]

The antigen 85 complex of Mycobacterium tuberculosis consists of three abundantly secreted proteins. The recent characterization of a mycoloyltransferase activity associated in vitro with each of these antigens suggested that they are potentially important for the building of the unusual cell envelope of mycobacteria. To define the physiological role of these proteins, the gene coding for antigen 85C was inactivated by transposon mutagenesis. The resulting mutant was shown to transfer 40% fewer mycolates to the cell wall with no change in the types of mycolates esterifying arabinogalactan or in the composition of non-covalently linked mycolates. As a consequence, the diffusion of the hydrophobic chenodeoxycholate and the hydrophilic glycerol, but not that of isoniazid, was found to be much faster through the cell envelope of the mutant than that of the parent strain. Taken together, these data demonstrate that: (i) antigen 85C is involved directly or indirectly in the transfer of mycolates onto the cell wall of the whole bacterium; (ii) the enzyme is not specific for a given type of mycolate; and (iii) the cell wall-linked mycolate layer may represent a barrier for the diffusion of small hydrophobic and hydrophilic molecules. [ABSTRACT FROM AUTHOR]

A luminescence-based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand-mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacteium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents. [ABSTRACT FROM AUTHOR]

Autophagy is an important innate immune defense mechanism that controls Mycobacterium tuberculosis (Mtb) growth inside macrophages. Autophagy machinery targets Mtb-containing phagosomes via xenophagy after damage to the phagosomal membrane due to the Type VII secretion system Esx-1 or via LC3-associated phagocytosis without phagosomal damage. Conversely, Mtb restricts autophagy-related pathways via the production of various bacterial protein factors. Although bacterial lipids are known to play strategic functions in Mtb pathogenesis, their role in autophagy manipulation remains largely unexplored. Here, we report that the lipid virulence factors sulfoglycolipids (SLs) and phthiocerol dimycocerosates (DIMs) control autophagy-related pathways through distinct mechanisms in human macrophages. Using knock-out and knock-in mutants of Mtb and Mycobacterium bovis BCG (Bacille Calmette Guerin) and purified lipids, we found that (i) Mtb mutants with DIM and SL deficiencies promoted functional autophagy via an MyD88-dependent and phagosomal damage-independent pathway in human macrophages; (ii) SLs limited this pathway by acting as TLR2 antagonists; (iii) DIMs prevented phagosomal damage-independent autophagy while promoting Esx-1-dependent xenophagy; (iv) and DIMs, but not SLs, limited the acidification of LC3-positive Mtb compartments. In total, our study reveals an unexpected and intricate role for Mtb lipid virulence factors in controlling autophagy-related pathways in human macrophages, thus providing further insight into the autophagy manipulation tactics deployed by intracellular bacterial pathogens. [ABSTRACT FROM AUTHOR]