SummaryInfection 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.

Background: Biosynthesis and functions of Mycobacterium marinum lipooligosaccharides (LOSs) remain elusive. Results: M. marinum mutants expressing various LOS profiles were generated and used to infect macrophages and amoebae. Conclusion: Deep LOS mutants are more efficiently phagocytosed than those lacking only LOS-IV. Significance: Three novel biosynthetic genes and the effect of the LOS content in modulating uptake by phagocytes are reported.

SummarySusceptibility 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.

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.

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.

bThe 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. The absence of novel effective antituberculosis therapy remains a significant worldwide public health threat due to the rapid development of multidrug-resistant and extensively drug-resistant strains (1, 2). The development of potent chemotherapeutic alternatives is crucial to preventing future epidemics of this insidious and often fatal form of the disease. New drugs to treat tuberculosis are urgently needed, yet the pace of new drug development has been slow. One approach to finding new agents is to define the pharmacological target(s) of currently used drugs and develop new therapeutic analogues that possess greater potency than the original molecules against Mycobacterium tuberculosis.Isoxyl (ISO; thiocarlide, 4,4=-diisoamythio-carbanilide) began to be used clinically to treat tuberculosis in the 1960s. We and others have shown that this thiourea derivative is a prodrug that must be activated by the mycobacterial monooxygenase EthA, a common activator of most thiocarbamide-containing drugs, including ethionamide and thiacetazone (3-5). In an early study, ISO was shown to inhibit the synthesis of both mycolic acids and free fatty acids in Mycobacterium bovis BCG (6). It was later reported that ISO displays potent activity against other slow-and fast-growing species of Mycobacterium, including multidrug-resistant clinical isolates of M. tuberculosis, by altering the biosynthesis of all types of mycolic acids and the long-chain fatty acids that are an important component of the mycobacterial cell wall and also alters the synthesis of shorter-chain fatty acids (7). The main effect of ISO on fatty acid metabolism is the inhibition of oleic acid biosynthesis by specifically targeting the membraneassociated stearoyl-coenzyme A (CoA) desaturase DesA3 (Rv3229c) (8). Sterculic acid, a known inhibitor of membraneassociated ⌬9 desaturases (8, 9), emulated the effect of ISO on oleic acid synthesis but did not inhibit mycolic acid synthesis (8), suggesting that the two effects of ISO were unrelated. Moreover, the fact that some ISO derivatives were found to affect mainly mycolic acid synthesis while having lost the capacity to inhibit oleic acid production supports the view that ISO has at least an additional target that is directly or indirectly related to the mycolic acid biosynthetic pathway (7).With the goal of elucidating the molecular mechanism of action of ISO inhibition on mycolic acid synthesis, genetic and biochemical studies of defined strains of M. tuberculosis were undertaken. We report here that components of the dehy...

Mature CD4؉ and CD8 ؉ T lymphocytes are believed to build and express essentially identical surface ␣␤ T-cell receptor-CD3 (TCR⅐CD3) complexes. However, TCR⅐CD3 expression has been shown to be more impaired in CD8 ؉ cells than in CD4؉ cells when CD3␥ is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR⅐CD3 complex in human CD3␥-deficient CD8؉ and CD4 ؉ T cells. The results indicated that the membrane TCR⅐CD3 complex of CD8 ؉ T lymphocytes was conformationally different from that of CD4 ؉ lymphocytes in the absence of CD3␥. In addition, CD8؉ , but not CD4 ؉ , CD3␥-deficient T lymphocytes were shown to contain abnormally glycosylated TCR␤ proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCR␣). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4؉ and CD8 ؉ T lymphocytes in the intracellular control of ␣␤TCR⅐CD3 assembly, maturation, or transport that are revealed when CD3␥ is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.Mature ␣␤ T lymphocytes recognize pathogen-derived peptides on antigen-presenting cells by means of the multimeric membrane protein ensemble termed the T-cell receptor (TCR) 1 ⅐CD3 complex. This TCR⅐CD3 complex includes two clonally distributed variable chains that directly interact with antigens (TCR␣ and TCR␤) and four invariant polypeptides that regulate assembly and signal transduction (CD3␥, CD3␦, CD3⑀, and ) (1). The assembly of complete TCR⅐CD3⅐ complexes takes place in a highly ordered manner within the endoplasmic reticulum: first CD3 chains, then TCR chains, and finally chains. Further conformational maturation, including carbohydrate processing, occurs in the Golgi apparatus before exportation of mature complexes to the T cell surface. The biochemical machinery involved in the assembly, processing, and exportation of TCR⅐CD3 complexes is assumed to be shared by all ␣␤ T-lineage cells. Thus, CD4ϩ and CD8 ϩ are believed to build biochemically and conformationally identical antigen receptors, although differences in the numbers that reach or remain at the cell surface have been noted (2). Therefore, the lack of any CD3 chain would be expected to affect to a similar extent the assembly and exportation of TCR⅐CD3 complexes by mature CD4 ϩ and CD8 ϩ T cells. However, this was not the case in several murine and human CD3 deficiencies (reviewed in Ref.3). In particular, it has been consistently shown that in the absence of CD3␥ or CD3␦, TCR⅐CD3 expression (or conformation) is more impaired in mature peripheral CD8 ϩ cells than in their CD4 ϩ counterparts, both in human and in murine deficiencies (3-7). Three other observations suggested the existence of CD8 ϩ cell-specific defects in human CD3␥ deficiency: first, the proband died after a viral infection (a cytolytic T-celldependent function) despite normal antibody responses (helper T-cell-dependent) (8...

The present study was performed in order to analyze whether T‐cell receptor (TCR)/CD3 assembly, intracellular transport and surface expression are carried in a similar way in αβ‐and γδ‐T cells. By means of optimal immunoprecipitation conditions with 35S‐methionine/cysteine‐ or biotin‐labelled TCR/CD3 proteins from αβ‐ or γδ‐T‐lymphoma‐cell lines, as well as TCRγδ cDNA transfectants, it was found that CD3δ chains associate less strongly with TCRγδ heterodimers compared to TCRαβ heterodimers. This preferential reactivity of CD3δ chains appears to be structural and not owing to differences in γδ‐ versus αβ‐T‐cell intracellular environments. Our results are in accordance firstly, with data from CD3δ‐deficient mice, which have γδ‐T cells but no αβ‐T cells, secondly with the suggested role of CD3δ chains in the positive selection of αβ‐T cells, a process apparently not followed by γδ‐T cells, and lastly with the differential roles of CD3δ chains versus CD3γ chains, explaining the maintenance of two CD3δ and CD3γ genes after the duplication from a CD3δ/γ gene present in avians. The impaired reactivity of CD3δ chains with TCRγδ heterodimers seems to be owing to a less efficient association with TCRγ chains. In contrast, CD3δ chains interact as strongly with TCRδ chains as do CD3γ chains with both TCRγ and TCRδ chains. These data may explain, at the molecular levels, why surface TCR/CD3 expression levels are impaired in γδ‐T cells from CD3γ‐deficient mice but not from CD3δ‐deficient mice.