Lipoteichoic Acid in Streptomyces hygroscopicus: Structural Model and Immunomodulatory Activities

Gram positive bacteria produce cell envelope macroamphiphile glycopolymers, i.e. lipoteichoic acids or lipoglycans, whose functions and biosynthesis are not yet fully understood. We report for the first time a detailed structure of lipoteichoic acid isolated from a Streptomyces species, i.e. Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387T. Chemical, MS and NMR analyses revealed a polyglycerolphosphate backbone substituted with α-glucosaminyl and α-N-acetyl-glucosaminyl residues but devoid of any amino-acid substituent. This structure is very close, if not identical, to that of the wall teichoic acid of this organism. These data not only contribute to the growing recognition that lipoteichoic acid is a cell envelope component of Gram positive Actinobacteria but also strongly support the recently proposed hypothesis of an overlap between the pathways of lipoteichoic acid and wall teichoic acid synthesis in these bacteria. S. hygroscopicus lipoteichoic acid induced signalling by human innate immune receptor TLR2, confirming its role as a microbe-associated molecular pattern. Its activity was partially dependant on TLR1, TLR6 and CD14. Moreover, it stimulated TNF-α and IL-6 production by a human macrophage cell line to an extent similar to that of Staphylococcus aureus lipoteichoic acid. These results provide new clues on lipoteichoic acid structure/function relationships, most particularly on the role of the polyglycerolphosphate backbone substituents

Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan

Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-14C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM

Lipoglycans Contribute to Innate Immune Detection of Mycobacteria

Innate immune recognition is based on the detection, by pattern recognition receptors (PRRs), of molecular structures that are unique to microorganisms. Lipoglycans are macromolecules specific to the cell envelope of mycobacteria and related genera. They have been described to be ligands, as purified molecules, of several PRRs, including the C-type lectins Mannose Receptor and DC-SIGN, as well as TLR2. However, whether they are really sensed by these receptors in the context of a bacterium infection remains unclear. To address this question, we used the model organism Mycobacterium smegmatis to generate mutants altered for the production of lipoglycans. Since their biosynthesis cannot be fully abrogated, we manipulated the biosynthesis pathway of GDP-Mannose to obtain some strains with either augmented (∼1.7 fold) or reduced (∼2 fold) production of lipoglycans. Interestingly, infection experiments demonstrated a direct correlation between the amount of lipoglycans in the bacterial cell envelope on one hand and the magnitude of innate immune signaling in TLR2 reporter cells, monocyte/macrophage THP-1 cell line and human dendritic cells, as revealed by NF-κB activation and IL-8 production, on the other hand. These data establish that lipoglycans are bona fide Microbe-Associated Molecular Patterns contributing to innate immune detection of mycobacteria, via TLR2 among other PRRs

Immunological hyporesponsiveness in tuberculosis: The role of mycobacterial glycolipids

Glycolipids constitute a major part of the cell envelope of Mycobacterium tuberculosis (Mtb). They are potent immunomodulatory molecules recognized by several immune receptors like pattern recognition receptors such as TLR2, DC-SIGN and Dectin-2 on antigen-presenting cells and by T cell receptors on T lymphocytes. The Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic relatives, phosphatidylinositol mannosides (PIMs) and lipomannan (LM), as well as other Mtb glycolipids, such as phenolic glycolipids and sulfoglycolipids have the ability to modulate the immune response, stimulating or inhibiting a pro-inflammatory response. We explore here the downmodulating effect of Mtb glycolipids. A great proportion of the studies used in vitro approaches although in vivo infection with Mtb might also lead to a dampening of myeloid cell and T cell responses to Mtb glycolipids. This dampened response has been explored ex vivo with immune cells from peripheral blood from Mtb-infected individuals and in mouse models of infection. In addition to the dampening of the immune response caused by Mtb glycolipids, we discuss the hyporesponse to Mtb glycolipids caused by prolonged Mtb infection and/or exposure to Mtb antigens. Hyporesponse to LAM has been observed in myeloid cells from individuals with active and latent tuberculosis (TB). For some myeloid subsets, this effect is stronger in latent versus active TB. Since the immune response in individuals with latent TB represents a more protective profile compared to the one in patients with active TB, this suggests that downmodulation of myeloid cell functions by Mtb glycolipids may be beneficial for the host and protect against active TB disease. The mechanisms of this downmodulation, including tolerance through epigenetic modifications, are only partly explored

Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives.

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection

Detection of Mycobacterium tuberculosis in Sputum by Gas Chromatography-Mass Spectrometry of Methyl Mycocerosates Released by Thermochemolysis

Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test

Ultralong C100 Mycolic Acids Support the Assignment of Segniliparus as a New Bacterial Genus

Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane

Interferon-α-Conditioned Human Monocytes Combine a Th1-Orienting Attitude with the Induction of Autologous Th17 Responses: Role of IL-23 and IL-12

IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α

Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis

The Role of the Novel Exopolyphosphatase MT0516 in Mycobacterium tuberculosis Drug Tolerance and Persistence

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions