Biophysical Interaction Landscape of Mycobacterial Mycolic Acids and Phenolic Glycolipids with Host Macrophage Membranes.

Lipidic adjuvant formulations consisting of immunomodulatory mycobacterial cell wall lipids interact with host cells following administration. The impact of this cross-talk on the host membrane's structure and function is rarely given enough consideration but is imperative to rule out nonspecific perturbation underlying the adjuvant. In this work, we investigated changes in the plasma membranes of live mammalian cells after exposure to mycobacterial mycolic acid (MA) and phenolic glycolipids, two strong candidates for lipidic adjuvant therapy. We found that phenolic glycolipid 1 softened the plasma membrane, lowering membrane tension and stiffness, but MA did not significantly change the membrane characteristics. Further, phenolic glycolipid 1 had a fluidizing impact on the host plasma membrane, increasing the fluidity and the abundance of fluid-ordered-disordered coexisting lipid domains. Notably, lipid diffusion was not impacted. Overall, MA and, to a lesser extent, phenolic glycolipid 1, due to minor disruption of host cell membranes, may serve as appropriate lipids in adjuvant formulations.

Biomarcadores en tuberculosis / Biomarkers in tuberculosis

Resumen Los métodos diagnósticos clásicos para la tuberculosis son de baja sensibilidad o son muy lentos en la obtención de resultados (baciloscopía, cultivo de Koch). De ahí nace la necesidad de nuevos métodos diagnósticos para esta enfermedad. Los biomarcadores surgen como una opción a esta problemática, con un buen rendimiento diagnóstico, costo y accesibilidad. Ellos permiten identificar la respuesta inflamatoria y/o metabólica del huésped, extrapolando la presencia de Mycobacterium tuberculosis; o identifican moléculas propias del patógeno. En la presente revisión se describen biomarcadores que presentan un buen rendimiento diagnóstico basados en metodologías de investigación de alto nivel (estudio de cohortes, prospectivos, muestreo consecutivo o aleatorizado, comparación de rendimiento diagnóstico frente a cultivo). Es necesario el desarrollo de estas nuevas técnicas con el fin de realizar el diagnóstico precoz de la enfermedad y lograr así su tan ansiada eliminación.

The classical laboratory diagnostic methods for tuberculosis have a low sensitivity or take a long time to know their results. New methods are underway. Biomarkers are a good option to improve our diagnostic approach to this disease. They have good performance, low cost and accessibility. They identify a patient's inflammatory or metabolic response to Mycobacterium Tuberculosis or identifies molecules that are typical of the pathogen. In this paper we sum up the biomarkers with a good diag-nostic performance described in well design investigations. Early diagnosis with these new techniques should contribute to the elimination of the disease.

Catenulobactins A and B, Heterocyclic Peptides from Culturing Catenuloplanes sp. with a Mycolic Acid-Containing Bacterium.

The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a mycolic acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells.

[Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Chojalactones A-C, cytotoxic butanolides isolated from Streptomyces sp. cultivated with mycolic acid containing bacterium.

The soil-derived bacterium, Streptomyces sp. CJ-5, was cocultured with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. The combined culture method significantly enhanced the production of the secondary metabolites in Streptomyces sp. CJ-5, leading to the isolation of three novel butanolide chojalactones A-C (1-3), with unusual γ-butyrolactone scaffolds. The complete structures, including the absolute configurations of 1-3, were determined based on spectroscopic data and total syntheses. In methylthiazole tetrazolium (MTT) assays, 1 and 2 showed moderate cytotoxicity against P388 cells.

Mycobacterium hippocampi sp. nov., a rapidly growing scotochromogenic species isolated from a seahorse with tail rot.

A Gram-positive, aerobic, non-motile, non-sporulating, acid-fast, and rod-shaped bacterium (BFLP-6(T)), previously isolated from a seahorse (Hippocampus guttulatus) with tail rot, was studied using a polyphasic taxonomic approach. Growth occurred at 15-35 °C (optimum 25 °C), at pH 5.0-10.0 (optimum pH 7.0) and at NaCl concentrations between 0 and 6 % (w/v). The G+C content of DNA was 66.7 mol%. The predominant fatty acids were C(18:1) ω9c, C(16:0) and C(16:1) ω6c. A mycolic acid pattern of alpha-mycolates and keto-mycolates was detected. Analysis of concatenated sequences (16S rRNA, rpoB, ssrA and tuf genes), and chemotaxonomic and phenotypic features indicated that strain BFLP-6(T) represents a novel species within the genus Mycobacterium, for which the name Mycobacterium hippocampi sp. nov. is proposed. The type strain is BFLP-6(T) (=DSM 45391(T) =LMG 25372(T)).

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis.

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen's persistence.

Characteristics of Mycobacterium smegmatis J15cs strain lipids.

Mycobacterium smegmatis is a rapidly growing, non-pathogenic mycobacterium, and M. smegmatis strain mc(2)155 in particular has been used as a tool for molecular analysis of mycobacteria because of its high rate of transformation. We examined another strain, M. smegmatis J15cs, which has the advantage of surviving for six days in murine macrophages. The J15cs strain produces a rough dry colony, and we hypothesized that the long survival of the J15cs strain was correlated with its cell wall components. Therefore, the lipid compositions of these two strains were compared. The subclasses and carbon species of the mycolic acids were very similar, and the major glycolipids and phospholipids were expressed in both strains. However, apolar glycopeptidolipids were deleted only in the J15cs strain. The presence of apolar glycopeptidolipids gives the cell wall a different structure. Moreover, the apolar glycopeptidolipids were recognized by macrophages via toll-like receptor 2, but not 4. We concluded that the absence of apolar glycopeptidolipids is a definitive feature of the J15cs strain, and affects its morphology and survival in host cells.

Biochemical disclosure of the mycolate outer membrane of Corynebacterium glutamicum.

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective ΔAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic ß-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis.

'Dr Granville's mummy' was described to the Royal Society of London in 1825 and was the first ancient Egyptian mummy to be subjected to a scientific autopsy. The remains are those of a woman, Irtyersenu, aged about 50, from the necropolis of Thebes and dated to about 600 BC. Augustus Bozzi Granville (1783-1872), an eminent physician and obstetrician, described many organs still in situ and attributed the cause of death to a tumour of the ovary. However, subsequent histological investigations indicate that the tumour is a benign cystadenoma. Histology of the lungs demonstrated a potentially fatal pulmonary exudate and earlier studies attempted to associate this with particular disease conditions. Palaeopathology and ancient DNA analyses show that tuberculosis was widespread in ancient Egypt, so a systematic search for tuberculosis was made, using specific DNA and lipid biomarker analyses. Clear evidence for Mycobacterium tuberculosis complex DNA was obtained in lung tissue and gall bladder samples, based on nested PCR of the IS6110 locus. Lung and femurs were positive for specific M. tuberculosis complex cell-wall mycolic acids, demonstrated by high-performance liquid chromatography of pyrenebutyric acid-pentafluorobenzyl mycolates. Therefore, tuberculosis is likely to have been the major cause of death of Irtyersenu.

Helcobacillus massiliensis gen. nov., sp. nov., a novel representative of the family Dermabacteraceae isolated from a patient with a cutaneous discharge.

Gram-positive, non-spore-forming rods (strain 6401990T), isolated from a human cutaneous discharge were subjected to a polyphasic taxonomy study. The only respiratory quinone was MK-7 and the major fatty acids were anteiso-C15:0 (34.3%), anteiso-C17:0 (18.7%) and iso-C16:0 (18.6%). Mycolic acids were not present. Polar lipids present were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and unidentified glycolipids. The isomer of diaminopimelic acid identified was meso-diaminopimelic acid, and the analysis of whole-cell sugars showed the presence of high amounts of galactose, ribose and some glucose. The G+C content of strain 6401990T was 68.6%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed 95.1% similarity with Dermabacter hominis. On the basis of phenotypic data and phylogenetic inference, it is proposed that this strain represents a novel species in a new genus of the family Dermabacteraceae, for which the name Helcobacillus massiliensis gen. nov., sp. nov. is proposed. The type strain is 6401990T (CSUR P17T=CIP 109418T=CCUG 53859T).

Gordonia cholesterolivorans sp. nov., a cholesterol-degrading actinomycete isolated from sewage sludge.

The taxonomic position of the cholesterol-degrading strain Chol-3(T), isolated from a sewage sludge sample, was clarified using a polyphasic taxonomic approach. Phylogenetic analysis of its 16S rRNA gene sequence, whole-cell fatty acid profile and mycolic acid composition revealed that this isolate is a member of the genus Gordonia with the species Gordonia sihwensis, G. hydrophobica and G. shandongensis being the nearest phylogenetic neighbours. The results of DNA-DNA hybridization against its phylogenetically closest neighbours as well as the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-3(T) from the other Gordonia species with validly published names. Strain Chol-3(T) therefore merits recognition as a member of a novel species within the genus Gordonia, for which the name Gordonia cholesterolivorans sp. nov. is proposed. The type strain is Chol-3(T) (=CECT 7408(T) =DSM 45229(T)).

Nocardia speluncae sp. nov., isolated from a cave.

The taxonomic status of a mycolic acid-containing actinomycete, isolated from a natural cave on Jeju Island, Republic of Korea, was investigated by means of a polyphasic approach. The isolate, designated strain N2-11(T), produced yellow- to orange-coloured vegetative hyphae and white- to pinkish white-coloured aerial mycelia, both of which fragmented into irregular rod-shaped elements. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the organism belonged to the family Nocardiaceae, occupying a distinct position between Nocardia harenae and a Nocardia carnea cluster. The results of chemotaxonomic analyses were consistent with the affiliation of the organism with the genus Nocardia. On the basis of 16S rRNA gene sequence similarities, the closest phylogenetic neighbours were the type strains of N. carnea (98.3 %), Nocardia flavorosea (98.0 %), Nocardia sienata (97.9 %) and Nocardia testacea (97.8 %), but the organism could be clearly distinguished from its phylogenetic relatives with reference to a broad range of physiological markers. On the basis of phenotypic and molecular genetic data presented in this study, strain N2-11(T) represents a novel species of the genus Nocardia, for which the name Nocardia speluncae sp. nov. is proposed. The type strain is N2-11(T) (=JBRI 2006(T) =KCTC 19223(T) =DSM 45078(T)).

Rhodococcus qingshengii sp. nov., a carbendazim-degrading bacterium.

A Gram-positive, aerobic, non-motile, mesophilic strain, djl-6(T), able to degrade carbendazim, was isolated from a carbendazim-contaminated soil sample from Jiangsu province, China. The taxonomic position of this isolate was analysed by using a polyphasic approach. Chemotaxonomic analysis including peptidoglycan type, diagnostic sugar composition, fatty acid profile, menaquinones, polar lipids and mycolic acids showed that the characteristics of strain djl-6(T) were in good agreement with those of the genus Rhodococcus. DNA-DNA hybridization showed that it had low genomic relatedness with Rhodococcus baikonurensis DSM 44587(T) (31.8 %), Rhodococcus erythropolis DSM 43066(T) (23.8 %) and Rhodococcus globerulus DSM 43954(T) (17.7 %), the three type strains to which strain djl-6(T) was most closely related based on 16S rRNA gene sequence analysis (99.78, 99.25 and 98.91 % similarity, respectively). Based on the phenotypic properties and DNA-DNA hybridization data, strain djl-6(T) (=CGMCC 1.6580(T) =KCTC 19205(T)) is proposed as the type strain of a novel Rhodococcus species, Rhodococcus qingshengii sp. nov.

Rhodococcus kyotonensis sp. nov., a novel actinomycete isolated from soil.

A polyphasic study was undertaken to establish the taxonomic position of an isolate, strain DS472(T), from soil in Kyoto, Japan. Phylogenetic analysis, based on the 16S rRNA gene sequences, revealed that this strain constitutes a new subline within the genus Rhodococcus, with Rhodococcus yunnanensis YIM 70056(T) and Rhodococcus fascians DSM 20669(T) as its nearest phylogenetic neighbours (98.2 and 97.8 % sequence similarity, respectively). DNA-DNA hybridization experiments revealed 36 and 29 % relatedness between the isolate and its phylogenetic relatives, R. yunnanensis and R. fascians, respectively. Chemotaxonomic characteristics, including the major quinone MK-8(H(2)), predominant fatty acids C(16 : 0), C(18 : 1)omega9c and 10-methyl C(18 : 0), the presence of cell-wall chemotype IV and mycolic acids, were consistent with the properties of members of the genus Rhodococcus. The DNA G+C content was 64.5 mol%. On the basis of both phenotypic and genotypic evidence, strain DS472(T) represents a novel species of the genus Rhodococcus, for which the name Rhodococcus kyotonensis sp. nov. is proposed. The type strain is strain DS472(T) (=IAM 15415(T)=CCTCC AB206088(T)).

Terrabacter aerolatus sp. nov., isolated from an air sample.

A Gram-positive, strictly aerobic, motile, rod- or coccoid-shaped bacterium, strain 5516J-36(T), was isolated from an air sample from Jeju region, Korea, and its taxonomic position was investigated by a polyphasic approach. The organism grew optimally at 30 degrees C and pH 7.0-8.0. Comparative 16S rRNA gene sequencing studies demonstrated that this strain was highly related phylogenetically to Terrabacter terrae PPLB(T) and Terrabacter tumescens DSM 20308(T), showing 98.9 % sequence similarity to both strains. However, the DNA-DNA reassociation values between 5516J-36(T) and the type strains of Terrabacter terrae and Terrabacter tumescens were low (51 and 48 %, respectively). The peptidoglycan type was A3gamma, the predominant menaquinone was MK-8(H(4)), the polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and an unidentified phosphoglycolipid and the whole-cell sugars were glucose, ribose, rhamnose, xylose and galactose. Mycolic acids were absent. The major fatty acids (>5 % of total fatty acids) were iso-C(15 : 0), iso-C(16 : 0), iso-C(14 : 0), iso-C(17 : 0) and anteiso-C(15 : 0). The DNA G+C content was 71.7 mol%. On the basis of the above data, it is proposed that strain 5516J-36(T) represents a novel species, Terrabacter aerolatus sp. nov. The type strain of Terrabacter aerolatus is 5516J-36(T) (=KACC 20556(T) =DSM 18562(T)).

Study on the cell wall skeleton derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105): establishment of preparation and analytical methods.

Mycobacterial cell walls have diverse adjuvant activities, and in particular, cell wall skeleton (CWS) of Mycobacterium bovis BCG has been expected as a drug for tumor immunotherapy. However, its molecular structure-biological activity relationship has not been fully elucidated despite more than 30 years of intensive research. Since it is important to secure purified CWS for such investigation, we established a preparation method of CWS from M. bovis BCG Tokyo 172 (SMP-105) and developed accurate, precise, and reliable analytical methods, based on previous reports. Furthermore, we confirmed that SMP-105 is composed of mycolic acids; arabinogalactan consisting of arabinose, galactose, and rhamnose; and peptidoglycan consisting of alanine, glutamic acid, diaminopimeric acid, muramic acid, glucosamine, and galactosamine. We also determined the levels of potential impurities that might be contaminated in the original bacterium or arise during the manufacturing process, such as glucose, mannose, non-constituted amino acids, as well as nucleic acid, trehaolse di-mycolate, and bacterial endotoxins. These results demonstrated that the prepared SMP-105 was of sufficient quality for research into the chemistry, bioactivity, and structure-activity relationship of CWS.

Williamsia serinedens sp. nov., isolated from an oil-contaminated soil.

The taxonomic status of a bacterium designated strain IMMIB SR-4(T) isolated from an oil-contaminated soil sample was characterized by using a polyphasic approach. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV, short-chain mycolic acids that co-migrated with those extracted from members of the genus Williamsia and that on pyrolysis GC produce C(16 : 0) and C(18 : 0) fatty acids, and dihydrogenated menaquinone with nine isoprene units as the predominant menaquinone. The generic assignment was confirmed by 16S rRNA gene sequence analysis. Comparative analysis of the 16S rRNA gene sequence showed that strain IMMIB SR-4(T) formed a distinct phyletic line within the genus Williamsia, displaying sequence similarities of 95.5-98.1 % with the type strains of recognized Williamsia species. Strain IMMIB SR-4(T) was distinguished from the type strains of recognized species of the genus Williamsia based on a set of phenotypic features. The genotypic and phenotypic data indicated that strain IMMIB SR-4(T) represents a novel species of the genus Williamsia, for which the name Williamsia serinedens sp. nov. is proposed. The type strain is IMMIB SR-4(T) (=DSM 45037(T)=CCUG 53151(T)).

Smaragdicoccus niigatensis gen. nov., sp. nov., a novel member of the suborder Corynebacterineae.

A polyphasic taxonomic approach was applied to determine the taxonomic position of a hydrocarbon-degrading actinomycete, strain Hou_blueT, which was isolated from soil samples collected from an oil spring in Niigata, Japan. The results of 16S rRNA and gyrB gene sequence comparisons indicated that strain Hou_blueT represented a novel lineage in the suborder Corynebacterineae. Colonies were malachite green-like in colour on 1/10 trypticase soy agar and the cell morphology was coccoid in all growth phases. The cell-wall diamino acid and sugar indicated chemotype IV and variation A1gamma. The sugars of the peptidoglycan were glycolated. The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and some unspecified glycolipids. The organism contained two novel cyclic forms of menaquinone, smaragdiquinone A-8(H4, omega-cycl) and smaragdiquinone B-8(H4, dicycl). The major fatty acids were cis-9-18 : 1 (34.46 %) and 16 : 0 (25.1 %). Small amounts of 10-methyl-branched fatty acids were also present (10-methyl-17 : 0, 0.17 %), but not tuberculostearic acid (10-methyl-18 : 0), which has been shown to be present in all nocardiae. Gas-chromatographic analysis of the mycolic acid revealed a carbon-chain length of C43-C49. The DNA G+C content was 63.7 mol%. On the basis of phenotypic and phylogenetic distinctness, the organism is proposed to represent a novel genus and species, Smaragdicoccus niigatensis gen. nov., sp. nov., with the type strain Hou_blueT (=MBIC 06267T=DSM 44881T).

Nocardia elegans sp. nov., a member of the Nocardia vaccinii clade isolated from sputum.

Two bacterial isolates from the sputa of a patient with a pulmonary infection were subjected to a polyphasic taxonomic study. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and mycolic acids consistent with the profile for the genus Nocardia. Comparative 16S rRNA gene sequencing showed that these isolates constitute a distinct subline within the genus Nocardia, displaying 99.6-95.5% sequence similarities with established species. However, DNA-DNA hybridization studies demonstrated unambiguously that the isolates are genealogically distinct from closely related species, namely Nocardia veterana and Nocardia africana, which show high levels of 16S rRNA sequence similarity (99.2 and 99.6% sequence similarity, respectively). On the basis of both phenotypic and phylogenetic evidence, it is proposed that these isolates be classified as a novel species of the genus Nocardia, for which the name Nocardia elegans sp. nov. is proposed. The type strain is IMMIB N-402(T) (=CCUG 50200(T)=CIP 108553(T)).