A truncated lipoglycan from mycobacteria with altered immunological properties

Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf–branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure–function relationship of TLR2 activation by mycobacterial lipoglycans

Test Characteristics of Urinary Lipoarabinomannan and Predictors of Mortality among Hospitalized HIV-Infected Tuberculosis Suspects in Tanzania.

Tuberculosis is the most common cause of death among patients with HIV infection living in tuberculosis endemic countries, but many cases are not diagnosed pre-mortem. We assessed the test characteristics of urinary lipoarabinomannan (LAM) and predictors of mortality among HIV-associated tuberculosis suspects in Tanzania. We prospectively enrolled hospitalized HIV-infected patients in Dar es Salaam, with ≥2 weeks of cough or fever, or weight loss. Subjects gave 2 mLs of urine to test for LAM using a commercially available ELISA, ≥2 sputum specimens for concentrated AFB smear and solid media culture, and 40 mLs of blood for culture. Among 212 evaluable subjects, 143 (68%) were female; mean age was 36 years; and the median CD4 count 86 cells/mm(3). 69 subjects (33%) had culture confirmation of tuberculosis and 65 (31%) were LAM positive. For 69 cases of sputum or blood culture-confirmed tuberculosis, LAM sensitivity was 65% and specificity 86% compared to 36% and 98% for sputum smear. LAM test characteristics were not different in patients with bacteremia but showed higher sensitivity and lower specificity with decreasing CD4 cell count. Two month mortality was 64 (53%) of 121 with outcomes available. In multivariate analysis there was significant association of mortality with absence of anti-retroviral therapy (p = 0.004) and a trend toward association with a positive urine LAM (p = 0.16). Among culture-negative patients mortality was 9 (75%) of 12 in LAM positive patients and 27 (38%) of 71 in LAM negative patients (p = 0.02). Urine LAM is more sensitive than sputum smear and has utility for the rapid diagnosis of culture-confirmed tuberculosis in this high-risk population. Mortality data raise the possibility that urine LAM may also be a marker for culture-negative tuberculosis

A Novel Sensitive Immunoassay Targeting the 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis

Identification and structural characterisation of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiAmutant

Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan, lipoarabinomannan (LAM). Herein, we show by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterised by single t-Araf residues linked to th

Analytic Treatment of Positronium Spin Splittings in Light-Front QED

We study the QED bound-state problem in a light-front hamiltonian approach. Starting with a bare cutoff QED Hamiltonian, $H_{_{B}}$, with matrix elements between free states of drastically different energies removed, we perform a similarity transformation that removes the matrix elements between free states with energy differences between the bare cutoff, $\Lambda$, and effective cutoff, \lam (\lam < \Lam). This generates effective interactions in the renormalized Hamiltonian, $H_{_{R}}$. These effective interactions are derived to order $\alpha$ in this work, with $\alpha \ll 1$. $H_{_{R}}$ is renormalized by requiring it to satisfy coupling coherence. A nonrelativistic limit of the theory is taken, and the resulting Hamiltonian is studied using bound-state perturbation theory (BSPT). The effective cutoff, \lam^2, is fixed, and the limit, 0 \longleftarrow m^2 \alpha^2\ll \lam^2 \ll m^2 \alpha \longrightarrow \infty, is taken. This upper bound on \lam^2 places the effects of low-energy (energy transfer below \lam) emission in the effective interactions in the $| e {\overline e} >$ sector. This lower bound on \lam^2 insures that the nonperturbative scale of interest is not removed by the similarity transformation. As an explicit example of the general formalism introduced, we show that the Hamiltonian renormalized to $O(\alpha)$ reproduces the exact spectrum of spin splittings, with degeneracies dictated by rotational symmetry, for the ground state through $O(\alpha^4)$. The entire calculation is performed analytically, and gives the well known singlet-triplet ground state spin splitting of positronium, $7/6 \alpha^2 Ryd$. We discuss remaining corrections other than the spin splittings and how they can be treated in calculating the spectrum with higher precision.Comment: 46 pages, latex, 3 Postscript figures included, section on remaining corrections added, title changed, error in older version corrected, cutoff placed in a windo

Tensor product structure of affine Demazure modules and limit constructions

Let \Lg be a simple complex Lie algebra, we denote by \Lhg the corresponding affine Kac--Moody algebra. Let $\Lambda_0$ be the additional fundamental weight of \Lhg. For a dominant integral \Lg--coweight \lam^\vee, the Demazure submodule V_{-\lam^\vee}(m\Lam_0) is a \Lg--module. For any partition of \lam^\vee=\sum_j \lam_j^\vee as a sum of dominant integral \Lg--coweights, the Demazure module is (as \Lg--module) isomorphic to \bigotimes_j V_{-\lam^\vee_j}(m\Lam_0). For the ``smallest'' case, \lam^\vee=\om^\vee a fundamental coweight, we provide for \Lg of classical type a decomposition of V_{-\om^\vee}(m\Lam_0) into irreducible \Lg--modules, so this can be viewed as a natural generalization of the decomposition formulas in \cite{KMOTU} and \cite{Magyar}. A comparison with the U_q(\Lg)--characters of certain finite dimensional U_q'(\Lhg)--modules (Kirillov--Reshetikhin--modules) suggests furthermore that all quantized Demazure modules V_{-\lam^\vee,q}(m\Lam_0) can be naturally endowed with the structure of a U_q'(\Lhg)--module. Such a structure suggests also a combinatorially interesting connection between the LS--path model for the Demazure module and the LS--path model for certain U_q'(\Lhg)--modules in \cite{NaitoSagaki}. For an integral dominant \Lhg--weight $\Lambda$ let V(\Lam) be the corresponding irreducible \Lhg--representation. Using the tensor product decomposition for Demazure modules, we give a description of the \Lg--module structure of V(\Lam) as a semi-infinite tensor product of finite dimensional \Lg--modules. The case of twisted affine Kac-Moody algebras can be treated in the same way, some details are worked out in the last section.Comment: 24 pages, in the current version we added the case of twisted affine Kac--Moody algebra