A characteristic phenolic glycolipid antigen from Mycobacterium haemophilum

A characteristic phenolic glycolipid was detected, by thin layer chromatography, in non-polar lipid extracts of nine representative strains of Mycobacterium haemophilum . The lipid was a specific antigen that reacted strongly with serum raised against whole cells of M. haemophilum. Sera from six other mycobacterial strains were tested but only that from Mycobacterium kansasii give a weak reaction.     

Elucidation of a protein-protein interaction network involved in Corynebacterium glutamicum cell wall biosynthesis as determined by bacterial two-hybrid analysis

Mycobacterium species have a highly complex and unique cell wall that consists of a large macromolecular structure termed the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex. This complex is essential for growth, survival and virulence of the human pathogen Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. The closely related species Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes and proteins involved in mAGP synthesis. This study examines the construction of a protein-protein interaction network for the major cell wall component arabinogalactan in C. glutamicum based on the use of a bacterial two-hybrid system. We have identified twenty-four putative homotypic and heterotypic protein interactions in vivo. Our results demonstrate an association between glycosyltransferases, GlfT1 and AftB, and interaction between the sub-units of decaprenylphosphoribose epimerase, DprE1 and DprE2. These analyses have also shown that AftB interacts with AftA, which catalyzes the addition of the first three arabinose units onto the galactan chain. Both AftA and AftB associate with other arabinofuranosyltransferases, including Emb and AftC, that elongate and branch the arabinan domain. Moreover, a number of proteins involved in arabinogalactan biosynthesis were shown to form dimers or multimers. These findings provide a useful recourse for understanding the biosynthesis and function of the mycobacterial cell wall, as well as providing new therapeutic targets.     

A hybrid of the transhydrogenases from Rhodospirillum rubrum and Mycobacterium tuberculosis catalyses rapid hydride transfer but not the complete, proton-translocating reaction

All transhydrogenases appear to have three components: dI, which binds NAD(H), and dIII, which binds NADP(H), protrude from the membrane, and dII spans the membrane. However, the polypeptide composition of the enzymes varies amongst species. The transhydrogenases of Mycobacterium tuberculosis and of Rhodospirillum rubrum have three polypeptides. Sequence analysis indicates that an ancestral three-polypeptide enzyme evolved into transhydrogenases with either two polypeptides (such as the Escherichia coli enzyme) or one polypeptide (such as the mitochondrial enzyme). The fusion steps in each case probably led to the development of an additional transmembrane helix. A hybrid transhydrogenase was constructed from the dI component of the M. tuberculosis enzyme and the dII and dIII components of the R. rubrum enzyme. The hybrid catalyses cyclic transhydrogenation but not the proton-translocating, reverse reaction. This shows that nucleotide-binding/release at the NAD(H) site, and hydride transfer, are fully functional but that events associated with NADP(H) binding/release are compromised. It is concluded that sequence mismatch in the hybrid prevents a conformational change between dI and dIII which is essential for the step accompanying proton translocation.     

Role of lipid trimming and CD1 groove size in cellular antigen presentation

Cellular CD1 proteins bind lipids that differ in length (C(12-80)), including antigens that exceed the capacity of the CD1 groove. This could be accomplished by trimming lipids to a uniform length before loading or by inserting each lipid so that it penetrates the groove to a varying extent. New assays to detect antigen fragments generated within human dendritic cells showed that bacterial antigens remained intact, even after delivery to lysosomes, where control lipids were cleaved. Further, recombinant CD1b proteins could bind and present C(80) lipid antigens using a mechanism that did not involve cellular enzymes or lipid cleavage, but was regulated by pH in the physiologic range. We conclude that endosomal acidification acts directly, rather than through enzymatic trimming, to insert lipids into CD1b. Lipids are loaded in an intact form, so that they likely protrude through a portal near the bottom of the groove, which represents an escape hatch for long lipids from mycobacterial pathogens.     

New lipophilic phthalimido- and 3-phenoxybenzyl sulfonates: inhibition of antigen 85C mycolyltransferase activity and cytotoxicity

Four new sulfonates were prepared as potential inhibitors of antigen 85C, a mycolyl transferase involved in the biosynthesis of the mycobacterial cell wall being designed on the basis of the proposed catalytic mechanism and antigen 85C crystal structure. The inhibitors contained a sulfonate moiety, 3-phenoxybenzyl alcohol or N-(hydroxyethyl)phthalimide as trehalose mimetics, and an alkyl chain of different length mimicking either the mycolate (alpha-chain or the mycolic acid (beta-branch. One compound displayed promising activity in a mycolyltransferase inhibition assay (compound 2b, IC50 = 4.3 microM). The two compounds containing a phthalimide moiety (compounds 3a and 3b) showed significant and selective cytotoxicity against the breast cancer cell line MDA-MB231.     

CD38 is required for the peripheral survival of immunotolerogenic CD4+ invariant NK T cells in nonobese diabetic mice

T cell-mediated autoimmune type-1 diabetes (T1D) in NOD mice partly results from this strain's numerical and functional defects in invariant NK T (iNKT) cells. T1D is inhibited in NOD mice treated with the iNKT cell superagonist alpha-galactosylceramide through a process involving enhanced accumulation of immunotolerogenic dendritic cells in pancreatic lymph nodes. Conversely, T1D is accelerated in NOD mice lacking CD38 molecules that play a role in dendritic cell migration to inflamed tissues. Unlike in standard NOD mice, alpha-galactosylceramide pretreatment did not protect the CD38-deficient stock from T1D induced by an adoptively transferred pancreatic beta cell-autoreactive CD8 T cell clone (AI4). We found that in the absence of CD38, ADP-ribosyltransferase 2 preferentially activates apoptotic deletion of peripheral iNKT cells, especially the CD4+ subset. Therefore, this study documents a previously unrecognized role for CD38 in maintaining survival of an iNKT cell subset that preferentially contributes to the maintenance of immunological tolerance.     

Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.     

Innate recognition of cell wall β-glucans drives invariant natural killer T cell responses against fungi

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to β-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of β-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.