Identification and characterization of the genes involved in glycosylation pathways of mycobacterial glycopeptidolipid biosynthesis

Glycopeptidolipids (GPLs) are major components present on the outer layers of the cell walls of several nontuberculous mycobacteria. GPLs are antigenic molecules and have variant oligosaccharides in mycobacteria such as Mycobacterium avium. In this study, we identified four genes (gtf1, gtf2, gtf3, and gtf4) in the genome of Mycobacterium smegmatis. These genes were independently inactivated by homologous recombination in M. smegmatis, and the structures of GPLs from each gene disruptant were analyzed. Thin-layer chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses revealed that the mutants Deltagtf1 and Deltagtf2 accumulated the fatty acyl-tetrapeptide core having O-methyl-rhamnose and 6-deoxy-talose as sugar residues, respectively. The mutant Deltagtf4 possessed the same GPLs as the wild type, whereas the mutant Deltagtf3 lacked two minor GPLs, consisting of 3-O-methyl-rhamnose attached to O-methyl-rhamnose of the fatty acyl-tetrapeptide core. These results indicate that the gtf1 and gtf2 genes are responsible for the early glycosylation steps of GPL biosynthesis and the gtf3 gene is involved in transferring a rhamnose residue not to 6-deoxy-talose but to an O-methyl-rhamnose residue. Moreover, a complementation experiment showed that M. avium gtfA and gtfB, which are deduced glycosyltransferase genes of GPL biosynthesis, restore complete GPL production in the mutants Deltagtf1 and Deltagtf2, respectively. Our findings propose that both M. smegmatis and M. avium have the common glycosylation pathway in the early steps of GPL biosynthesis but differ at the later stages.     

Effectiveness of BCG vaccination to aged mice

Background

The tuberculosis (TB) still increases in the number of new cases, which is estimated to approach 10 million in 2010. The number of aged people has been growing all over the world. Ageing is one of risk factors in tuberculosis because of decreased immune responses in aged people. Mycobacterium bovis Bacillus Calmette Guérin (BCG) is a sole vaccine currently used for TB, however, the efficacy of BCG in adults is still a matter of debate. Emerging the multidrug resistant Mycobacterium tuberculosis (MDR-TB) make us to see the importance of vaccination against TB in new light. In this study, we evaluated the efficacy of BCG vaccination in aged mice.

Results

The Th1 responses, interferon-γ production and interleukin 2, in BCG inoculated aged mice (24-month-old) were comparable to those of young mice (4- to 6-week-old). The protection activity of BCG in aged mice against Mycobacterium tuberculosis H₃₇Rv was also the same as young mice.

Conclusion

These findings suggest that vaccination in aged generation is still effective for protection against tuberculosis.     

Bacterial sphingophospholipids containing non-hydroxy fatty acid activate murine macrophages via Toll-like receptor 4 and stimulate bacterial clearance

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.     

IFN-γ plays an essential role in the pathogenesis of gastric lymphoid follicles formation caused by Helicobacter suis infection

In this study, we aimed to assess the role of helper T cells in the development of gastric lymphoid follicles induced by Helicobacter suis infection. C57BL/6J mice were orally inoculated with H. suis. Six weeks after infection, gastric lymphoid follicles were observed in the gastric mucosa by hematoxylin and eosin staining, and the number of follicles was increased throughout the infection period. An immunohistological examination showed that the lymphoid follicles were composed of B cells, CD4-positive helper T cells, and dendritic cells (DC). It was also revealed that the mRNA expression level of interferon-γ (IFN-γ) in the gastric mucosa was significantly increased at 12 weeks after infection. No gastric lymphoid follicles were detected in IFN-γ-deficient mice that had been infected with H. suis at 12 weeks after infection, although the development of lymphoid follicles in IL-4-deficient mice infected with H. suis was similar to that seen in the wild-type mice. In conclusion, IFN-γ, a Th1 cytokine, is deeply involved in the pathogenesis of gastric lymphoid follicles induced by H. suis infection, and it is suggested that CD4-positive T cells and DC aid in the expansion of gastric lymphoid follicles.     

The Mycobacterium avium complex gtfTB gene encodes a glucosyltransferase required for the biosynthesis of serovar 8-specific glycopeptidolipid

Mycobacterium avium complex (MAC) is one of the most common opportunistic pathogens widely distributed in the natural environment. The 28 serovars of MAC are defined by variable oligosaccharide portions of glycopeptidolipids (GPLs) that are abundant on the surface of the cell envelope. These GPLs are also known to contribute to the virulence of MAC. Serovar 8 is one of the dominant serovars isolated from AIDS patients, but the biosynthesis of serovar 8-specific GPL remains unknown. To clarify this, we compared gene clusters involved in the biosynthesis of several serovar-specific GPLs and identified the genomic region predicted to be responsible for GPL biosynthesis in a serovar 8 strain. Sequencing of this region revealed the presence of four open reading frames, three unnamed genes and gtfTB, the function of which has not been elucidated. The simultaneous expression of gtfTB and two downstream genes in a recombinant Mycobacterium smegmatis strain genetically modified to produce serovar 1-specific GPL resulted in the appearance of 4,6-O-(1-carboxyethylidene)-3-O-methyl-glucose, which is unique to serovar 8-specific GPL, suggesting that these three genes participate in its biosynthesis. Furthermore, functional analyses of gtfTB indicated that it encodes a glucosyltransferase that transfers a glucose residue via 1→3 linkage to a rhamnose residue of serovar 1-specific GPL, which is critical to the formation of the oligosaccharide portion of serovar 8-specific GPL. Our findings might provide a clue to understanding the biosynthetic regulation that modulates the biological functions of GPLs in MAC.     

Comparable studies of immunostimulating activities in vitro among Mycobacterium bovis bacillus Calmette–Guérin (BCG) substrains

During the serial passage of Mycobacterium bovis bacillus Calmette–Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. 'Early-shared strains' of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (α, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1β (IL-1β), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-α, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-γ (IFN-γ) than did 'late-shared strains' of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-α in the presence of IFN-γ was also observed by trehalose 6,6'-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that 'early-shared strains' are more potent immunostimulating agents than 'late-shared strains', which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.     

Different effects of novel mtDNA G3242A and G3244A base changes adjacent to a common A3243G mutation in patients with mitochondrial disorders

Two novel mitochondrial DNA base changes were identified at both sides of the 3243A > G mutation, the most common mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). One was a 3244G > A transition in a girl with MELAS. The other was a 3242G > A transition in a girl with a mitochondrial disorder without a MELAS phenotype. Although the two base changes were adjacent to the 3243A > G mutation, they had different effects on the clinical phenotype, muscle pathology, and respiratory chain enzyme activity. Investigations of the different effects of the 3244G > A and 3242G > A base changes may provide a better understanding of tRNA dysfunction in mitochondrial disorders.     

Identification of pyrrolo[2,3-d]pyrimidines as potent HCK and FLT3-ITD dual inhibitors

A series of novel pyrrolo[2,3-d]pyrimidines were synthesized by introducing 15 different amino acids to 7-cyclohexyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine. Compounds with potent activities against HCK and FLT3-ITD were evaluated in viability studies with acute myeloid leukemia cell line MV4-11. Our structure activity relationship analyses lead to the identification of compound 31, which exhibited potent HCK and FLT3-ITD inhibition and activity against the MV4-11 cell line.     

Essential role of satellite cells in the growth of rat soleus muscle fibers

Effects of gravitational loading or unloading on the growth-associated increase in the cross-sectional area and length of fibers, as well as the total fiber number, in soleus muscle were studied in rats. Furthermore, the roles of satellite cells and myonuclei in growth of these properties were also investigated. The hindlimb unloading by tail suspension was performed in newborn rats from postnatal day 4 to month 3 with or without 3-mo reloading. The morphological properties were measured in whole muscle and/or single fibers sampled from tendon to tendon. Growth-associated increases of soleus weight and fiber cross-sectional area in the unloaded group were approximately 68% and 69% less than the age-matched controls. However, the increases of number and length of fibers were not influenced by unloading. Growth-related increases of the number of quiescent satellite cells and myonuclei were inhibited by unloading. And the growth-related decrease of mitotically active satellite cells, seen even in controls (20%, P > 0.05), was also stimulated (80%). The increase of myonuclei during 3-mo unloading was only 40 times vs. 92 times in controls. Inhibited increase of myonuclear number was not related to apoptosis. The size of myonuclear domain in the unloaded group was less and that of single nuclei, which was decreased by growth, was larger than controls. However, all of these parameters, inhibited by unloading, were increased toward the control levels generally by reloading. It is suggested that the satellite cell-related stimulation in response to gravitational loading plays an essential role in the cross-sectional growth of soleus muscle fibers.     

Long distance mobility of male Japanese macaques evidenced by mitochondrial DNA

A 487-nucleotide sequence in the polymorphic D-loop region of matrilineally inherited mitochondrial DNA was compared in samples from 14 (9 adult males and 5 females or young) Japanese macaques (Macaca fuscata fuscata) of two troops at Gagyusan in Takahashi City, Okayama Prefecture, Japan. The majority (7 out of the sampled 9) of the adult males associated with the troops had nucleotide sequences of six distinct types, all of which were different from that of the Gagyusan troop females, whereas two young solitary males shared the same sequence. The present study exemplified a case in which the majority of adult males immigrated from outside the local population. Notably, two of the males were revealed to share the same sequence determined for a matriline 45 km away. Electronic Publication