A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence of Mycobacterium marinum in multiple hosts.

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C-terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail-forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane-defined vacuoles. Functional complementation studies indicated that the C-terminus, but not the N-terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non-pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis.

A new dehydratase conferring innate resistance to thiacetazone and intra-amoebal survival of Mycobacterium smegmatis.

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second-line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon-mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100-fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot-dog domains characteristic of dehydratase-like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis-Acanthamoeba co-cultures, we found that intra-amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.

Increased phagocytosis of Mycobacterium marinum mutants defective in lipooligosaccharide production: a structure-activity relationship study.

Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in ectotherms and is an occasional opportunistic human pathogen. In the environment, M. marinum also interacts with amoebae, which may serve as a natural reservoir for this microorganism. However, the description of mycobacterial determinants in the early interaction with macrophages or amoebae remains elusive. Lipooligosaccharides (LOSs) are cell surface-exposed glycolipids capable of modulating the host immune system, suggesting that they may be involved in the early interactions of M. marinum with macrophages. Herein, we addressed whether LOS composition affects the uptake of M. marinum by professional phagocytes. Mutants with various truncated LOS variants were generated, leading to the identification of several previously uncharacterized biosynthetic genes (wbbL2, MMAR_2321, and MMAR_2331). Biochemical and structural approaches allowed resolving the structures of LOS precursors accumulating in this set of mutants. These strains with structurally defined LOS profiles were then used to infect both macrophages and Acanthamoebae. An inverse correlation between LOS completeness and uptake of mycobacteria by phagocytes was found, allowing the proposal of three mutant classes: class I (papA4), devoid of LOS and highly efficiently phagocytosed; class II, accumulating only early LOS intermediates (wbbL2 and MMAR_2331) and efficiently phagocytosed but less than class I mutants; class III, lacking LOS-IV (losA, MMAR_2319, and MMAR_2321) and phagocytosed similarly to the control strain. These results indicate that phagocytosis is conditioned by the LOS pattern and that the LOS pathway used by M. marinum in macrophages is conserved during infection of amoebae.

Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis.

The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in Mycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

Fatty acyl chains of Mycobacterium marinum lipooligosaccharides: structure, localization and acylation by PapA4 (MMAR_2343) protein.

We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR_2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR_2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR_2343 gene. Disruption of MMAR_2343 resulted in a severe LOS breakdown, indicating that MMAR_2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly.

A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.

Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.

Temperature-dependent regulation of mycolic acid cyclopropanation in saprophytic mycobacteria: role of the Mycobacterium smegmatis 1351 gene (MSMEG_1351) in CIS-cyclopropanation of alpha-mycolates.

The cell envelope is a crucial determinant of virulence and drug resistance in Mycobacterium tuberculosis. Several features of pathogenesis and immunomodulation of host responses are attributable to the structural diversity in cell wall lipids, particularly in the mycolic acids. Structural modification of the alpha-mycolic acid by introduction of cyclopropane rings as catalyzed by the methyltransferase, PcaA, is essential for a lethal, persistent infection and the cording phenotype in M. tuberculosis. Here, we demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Interestingly, alpha-mycolic acid cyclopropanation was inducible in cultures grown at 25 degrees C. The growth temperature modulation of the cyclopropanating activity was determined by high resolution magic angle spinning NMR analyses on whole cells. In parallel, quantitative reverse transcription-PCR analysis showed that MSMEG_1351 gene expression is up-regulated at 25 degrees C compared with 37 degrees C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of alpha-mycolates. The functional equivalence of PcaA and MSMEG_1351 was established by cross-complementation in the MSMEG_1351 knock-out mutant and also in a DeltapcaA strain of Mycobacterium bovis BCG. Overexpression of MSMEG_1351 restored the wild-type mycolic acid profile and the cording phenotype in BCG. Although the biological significance of mycolic acid cyclopropanation in nonpathogenic mycobacteria remains unclear, it likely represents a mechanism of adaptation of cell wall structure and composition to cope with environmental factors.

Mycolic acid methyltransferase, MmaA4, is necessary for thiacetazone susceptibility in Mycobacterium tuberculosis.

Susceptibility of Mycobacterium tuberculosis to the second-line antitubercular drug thiacetazone (TAC) requires activation by the monoxygenase, EthA. Here, we report isolation of spontaneous mutants in Mycobacterium bovis BCG that are highly resistant to TAC, but carry a functional EthA. Unexpectedly, a majority of the TAC-resistant mutants lacked keto-mycolic acids, which are long-chain fatty acids associated with the cell wall and which contribute significantly to the physiopathology of tuberculosis. Predictably, causative mutations in the above mutants were in the gene encoding methyltransferase MmaA4, which is required for synthesis of keto- and methoxy-mycolic acids. Drug-resistant phenotype of the BCG mutants was reproduced in a mmaA4, but not in a mmaA3 null mutant of M. tuberculosis CDC1551. Susceptibility to TAC could be restored by complementation with a functional mmaA4 gene. Interestingly, overexpression of MmaA4 in M. bovis BCG made it more susceptible to TAC. We provide novel mechanistic insights into antitubercular drug activation by co-ordinated actions of EthA and MmaA4. This study is the first demonstration of the participation of an enzyme linked to the synthesis of oxygenated mycolates in a drug activation process in M. tuberculosis, and highlights the interplay between mycolic acid synthesis, drug activation and mycobacterial virulence.

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa, and identified mutants with attenuated virulence towards Dictyostelium. These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa.

Synergistic control of cellular adhesion by transmembrane 9 proteins.

The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface.

Dictyostelium discoideum transformation by oscillating electric field electroporation.

Dictyostelium discoideum has been used as a genetically tractable model organism to study many biological phenomena. High-efficiency transformation is a prerequisite for successful genetic screens such as mutant complementation, identification of suppressor genes, or insertional mutagenesis. Although exponential decay electroporation is the standard transformation technique for D. discoideum, its efficiency is relatively low and its reproducibility is weak. Here we optimized the oscillating electroporation technique for D. discoideum transformation and compared it to the exponential decay electroporation. A 20-fold increase in the efficiency was resproducibly achieved. This alternative electroporation technique should facilitate future genetic approaches in D. discoideum.

Overexpression of the KdpF membrane peptide in Mycobacterium bovis BCG results in reduced intramacrophage growth and altered cording morphology.

Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, such as sensor kinases. To date, regulatory membrane peptides have been completely overlooked in mycobacteria. The 30 amino-acid-long KdpF peptide, which is co-transcribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s) towards the KdpD sensor kinase. Herein, we showed by quantitative RT-PCR that the Mycobacterium bovis BCG kdpAB and kdpDE genes clusters are differentially induced in potassium-deprived broth medium or within infected macrophages. We have overexpressed the kdpF gene in M. bovis BCG to investigate its possible regulatory role and effect on mycobacterial virulence. Our results indicate that KdpF does not play a critical regulatory role on kdp genes expression despite the fact that KdpF interacts with the KdpD sensor kinase in a bacterial two-hybrid assay. However, overexpression of kdpF results in a significant reduction of M. bovis BCG growth in both murine and human primary macrophages, and is associated with a strong alteration of colonial morphology and impaired cording formation. To identify novel KdpF interactants, a mycobacterial library was screened using KdpF as bait in the bacterial two-hybrid system. This allowed us to identify members of the MmpL family of membrane proteins, known to participate in the biosynthesis/transport of various cell wall lipids, thus highlighting a possible link between KdpF and cell wall lipid metabolism. Taken together, these data suggest that KdpF overexpression reduces intramacrophage growth which may result from alteration of the mycobacterial cell wall.

Formation of multivesicular endosomes in Dictyostelium.

Multivesicular endosomes are present in virtually every eucaryotic cell, where they arise by intra-endosomal budding of the limiting endosomal membrane. Some genetic diseases such as Chediak-Higashi syndrome are characterized by enlarged membrane-filled endosomes. The same altered endosomal morphology can be observed in cells exposed to certain drugs, for example U18666A. The mechanisms involved are still poorly characterized, partially because this atypical budding event is particularly difficult to observe in mammalian cells. Taking advantage of the simplicity of the endosomal structure in Dictyostelium discoideum, we could visualize intraendosomal budding at the ultrastructural level. In this model organism, the drug U18666A was shown to stimulate intra-endosomal budding, while an inhibitor of PI 3-kinase activity was found to have no effect on this process. Inactivation of a Dictyostelium gene with similarity to the gene responsible for Chediak-Higashi syndrome did not alter the intra-endosomal budding or the accumulation of intra-endosomal membranes. Thus, although treatment with U18666A and inactivation of the Chediak-Higashi gene cause similar morphological defects in mammalian cells, observations in a different model reveal that their respective modes of action are different.