Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

Bacille Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis currently used as a vaccine against tuberculosis. Global distribution and propagation of BCG has contributed to the in vitro evolution of the vaccine strain and is thought to partially account for the different outcomes of BCG vaccine trials. Previous efforts by several molecular techniques effectively identified large sequence polymorphisms among BCG daughter strains, but lacked the resolution to identify smaller changes. In this study, we have used a NimbleGen tiling array for whole genome comparison of 13 BCG strains. Using this approach, in tandem with DNA resequencing, we have identified six novel large sequence polymorphisms including four deletions and two duplications in specific BCG strains. Moreover, we have uncovered various polymorphisms in the phoP-phoR locus. Importantly, these polymorphisms affect genes encoding established virulence factors including cell wall complex lipids, ESX secretion systems, and the PhoP-PhoR two-component system. Our study demonstrates that major virulence factors are different among BCG strains, which provide molecular mechanisms for important vaccine phenotypes including adverse effect profile, tuberculin reactivity and protective efficacy. These findings have important implications for the development of a new generation of vaccines.

by MDR strains that are also resistant to a fluoroquinolone and at least one second-line injectable agent (amikacin, kanamycin and/or capreomycin), caught the world's attention after an outbreak in KwaZulu-Natal, South Africa, where 52 of 53 infected patients died. 2 HIV co-infection was a contributing factor in most of these deaths, and indeed, a deadly association between HIV and TB has been known almost since the start of the HIV-epidemic. Of the 1.7 million people who died from TB in 2006, an estimated 200,000 were co-infected with HIV. 1 Because of these situations, effective approaches alternative to antibiotics are urgently needed for the control of TB.Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is currently the only available vaccine against TB. Since 1974, BCG vaccination has been included in the WHO Expanded Program on Immunization. 3 It is estimated that more than 3 billion individuals have been immunized with BCG and over 100 million doses of BCG are administered annually, making it the most widely used vaccine in humans. 3 Meta-analysis studies have confirmed that BCG protects children, providing >80% efficacy against severe forms of TB, including tuberculous meningitis and miliary TB. 4,5 In contrast, evidence for protection against pulmonary TB in adolescents and adults remains contentious as efficacy estimates from clinical trials, observational case control studies and contact studies range from 0 to 80%. 6,7 The reasons for the variable protective efficacy are unknown but several hypotheses have been proposed, including differences among the vaccine strains used in clinical studies, exposure of trial populations to environmental mycobacteria, nutritional or genetic differences in human populations, differences in trial methods, and variations among clinical M. tb strains. 8-13 These explanations are not mutually exclusive and all may contribute to the heterogeneity in vaccine efficacy.A key aspect of this issue may concern the nature of BCG attenuation. Although it is generally considered safe and has been used as a human vaccine since the 1920s, the mechanisms of BCG attenuation remain largely unknown. This is further complicated by the fact that BCG is not a single strain, but instead comprises a number of substrains that exhibit phenotypic and biochemical differences. 14 BCG strains also exhibit differences in residual virulence level. [15][16][17] However, side effects were often attributed to variations in the viability of vaccines during preparation procedures (e.g., freezedrying). 14 In other words, the observed differential virulence among Mycobacterium bovis Bacille Calmette-Guérin (BCG) was developed as an attenuated live vaccine for tuberculosis control nearly a century ago. Despite being the most widely used vaccine in human history, the mechanisms of attenuation of BCG remain poorly understood. BCG is not a single organism, but comprises a number of substrains that differ in genotypes and phenotypes. The impacts of these differences on BCG...

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes including cell wall biosynthesis and antibiotic resistance. However, the underlying molecular mechanisms remain unknown. In this article, we performed biochemical studies of Lsr2–DNA interactions and structure–function analysis of Lsr2. Analysis by atomic force microscopy revealed that Lsr2 has the ability to bridge distant DNA segments, suggesting that Lsr2 plays a role in the overall organization and compactness of the nucleoid. Mutational analysis identified critical residues and selection of dominant negative mutants demonstrated that both DNA binding and protein oligomerization are essential for the normal functions of Lsr2 in vivo. These results provide strong evidence that Lsr2 is a DNA bridging protein, which represents the first identification of such proteins in bacteria phylogenetically distant from the Enterobacteriaceae. DNA bridging by Lsr2 also provides a mechanism of transcriptional regulation by Lsr2.

c Phthiocerol dimycocerosates (PDIMs) and structurally related phenolic glycolipids (PGLs) are complex cell wall lipids unique to pathogenic mycobacteria. While these lipids have been extensively studied in recent years, there are conflicting reports on some aspects of their biosynthesis and on the role of PDIMs and especially PGLs in virulence of Mycobacterium tuberculosis. This has been complicated by the natural deficiency of PGLs in many clinical strains of M. tuberculosis and the frequent loss of PDIMs in laboratory M. tuberculosis strains. In this study, we isolated seven mutants of Mycobacterium marinum deficient in PDIMs and/or PGLs in which multiple genes of the PDIM/PGL biosynthetic locus were disrupted by transposon insertion. Zebrafish infection experiments showed that M. marinum strains lacking one or both of these lipids were avirulent, suggesting that both PDIMs and PGLs are required for virulence. We also found that these strains were hypersensitive to antibiotics and exhibited increased cell wall permeability. Our studies provide new insights into the biosynthesis of PDIMs/PGLs and may help us to understand the role of PDIMs and PGLs in M. tuberculosis virulence. P athogenic mycobacteria produce two structurally related, methyl-branched fatty acid-containing lipids called phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs). PDIMs and PGLs have long-chain fatty acid backbones consisting of 3-methoxy (or 3-keto, 3-hydroxy), 4-methyl, 9,11-dihydroxy glycols (phthiocerols) and p-glycosylated phenylglycols (glycosyl phenolphthiocerols), respectively, that are diesterified with di-, tri-, and tetramethyl-branched acyl chains (mycocerosates) (reviewed in reference 28). PDIMs have been identified in Mycobacterium tuberculosis, M. africanum, M. bovis, M. leprae, M. marinum, M. ulcerans, M. kansasii, M. haemophilum, M. microti, and M. gastri, all of which are pathogenic for humans or animals. PGLs are produced by the same set of pathogenic mycobacterial species, except that in M. tuberculosis only a subset of clinical isolates produces PGLs.The role of PDIMs in virulence was first suggested by two independent studies using signature-tagged transposon mutagenesis, which identified mutants of M. tuberculosis that were unable to either produce or properly localize PDIMs to the cell wall and demonstrated that these mutants were attenuated in animal models of infection (8,12,33). Since then, circumstantial evidence supporting a role for PDIMs in M. tuberculosis virulence has accumulated. The role of PGLs in M. tuberculosis virulence is less clear and is confounded by the fact that laboratory strains (H37Rv, Erdman) and many clinical isolates, including CDC1551 and MT103, are naturally deficient in PGL production due to a 7-basepair deletion in pks15/1, while some clinical isolates of the East Asian lineage have an intact pks15/1 gene and produce PGLs (31). Mutations of pks15/1 in M. tuberculosis HN878, a strain that produces both PDIMs and PGLs and exhibits a hypervirulent phenotype in infect...

Cerebral malaria is a leading cause of global morbidity and mortality. Interventions targeting the underlying pathophysiology of cerebral malaria may improve outcomes compared to treatment with antimalarials alone. Microvascular leak plays an important role in the pathogenesis of cerebral malaria. The angiopoietin (Ang)-Tie-2 system is a critical regulator of vascular function. We show that Ang-1 expression and soluble Tie-2 expression were associated with disease severity and outcome in a prospective study of Ugandan children with severe malaria and in a preclinical murine model of experimental cerebral malaria. Ang-1 was necessary for maintenance of vascular integrity and survival in a mouse model of cerebral malaria. Therapeutic administration of Ang-1 preserved blood-brain barrier integrity and, in combination with artesunate treatment, improved survival beyond that with artesunate alone. These data define a role for dysregulation of the Ang-Tie-2 axis in the pathogenesis of cerebral malaria and support the evaluation of Ang-Tie-2-based interventions as potential adjunctive therapies for treating severe malaria.

The significant increase of H(2)O(2) and reduction in antioxidant capacity in the EBC of lung cancer patients further support the concept of the disequilibrium between levels of oxidants and antioxidants in lung cancer, which leads to increased oxidative stress. These findings suggest oxidative stress is implicated in the development of lung cancer and may be an early marker of the disease.

To the Editors:The recent paper by ROSIAS et al. [1] reported that silicone and glass inner coatings were superior to the EcoScreen1 system (Erich Jaeger GmbH, Hochberg, Germany), aluminium, polypropylene and Teflon when measuring 8-isoprostane and albumin levels in exhaled breath condensate (EBC) [1][2][3]. This raises important issues in the ongoing debate about the optimal collection method for sampling airway biomarkers. We have complemented these important data by comparing the efficiency and reproducibility of EBC biomarkers collected by different devices in six healthy subjects.Four collection systems were compared in a randomised order: glass, siliconised glass, EcoScreen1 and RTube1 (Respiratory Research Inc., Charlottesville, VA, USA). Oxides of nitrogen (NO x ), total protein, mucin and pH were measured and assessed for reproducibility over 3 days [4,5].