Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis

Mycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin-binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope. Four different fbp genes are found in the genome of M. tuberculosis, but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown. We have shown previously that, in the heterologous host, Corynebacterium glutamicum, FbpA, FbpB and FbpC can all add mycoloyl residues to the cell wall arabinogalactan and that, in M. tuberculosis, the cell wall mycoloylation decreases by 40% when fbpC is knocked out. To investigate whether the remaining 60% mycoloylation came from the activity of FbpA and/or FbpB, fbpA- and fbpB-inactivated mutant strains were biochemically characterized and compared with the previously studied fbpC-disrupted mutant. Unexpectedly, both mutants produced normally mycoloylated cell walls. Overproduction of FbpA, FbpB or FbpC, but not FbpD, in the fbpC-inactivated mutant strain of M. tuberculosis restored both the cell wall-linked mycolate defect and the outer cell envelope permeability barrier property. These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps. In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC-inactivated mutant. Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan mycoloylation regions. Neither FbpA nor FbpB attaches mycoloyl residues to specific sites but, like FbpC, each enzyme transfers mycoloyl residues onto the four sites present in the arabinogalactan non-reducing end hexaarabinosides.     

Schistosomiasis,Soil-Transmitted Helminthiasis,and Sociodemographic Factors Influence Quality of Life of Adults in C?te d'Ivoire

Background

Burden of disease estimates are widely used for priority setting in public health and disability-adjusted life years are a powerful “currency” nowadays. However, disability weights, which capture the disability incurred by a typical patient of a certain condition, are fundamental to such burden calculation and their determination remains a widely debated issue.

Methodology

A cross-sectional epidemiological survey was conducted in the recently established Taabo health demographic surveillance system (HDSS) in south-central Côte d''Ivoire, to provide new, population-based evidence on the disability caused by schistosomiasis and soil-transmitted helminthiasis. Parasitological results from stool, urine, and blood examinations were juxtaposed to quality of life (QoL) questionnaire results from 187 adults. A multivariable linear regression model with stepwise backward elimination was used to identify significant associations, considering also sociodemographic characteristics obtained from the Taabo HDSS database.

Principal Findings

Prevalences for hookworm, Plasmodium spp., Trichuris trichiura, Schistosoma haematobium and Schistosoma mansoni were 39.0%, 18.2%, 2.7%, 2.1% and 2.1%, respectively. S. mansoni and T. trichiura infections of any intensity reduced the participants'' self-rated QoL by 16 points (95% confidence interval (CI): 4–29 points) and 13 points (95% CI: 1–24 points), respectively, on a scale from 0 (worst QoL) to 100 points (best QoL). The only other statistically significant effect was a 1-point (95% CI: 0.1–2 points) increase on the QoL scale per one unit increase in a calculated wealth index.

Conclusions/Significance

We found consistent and significant results on the negative effects of schistosomiasis and soil-transmitted helminthiasis on adults'' self-rated QoL, also when taking sociodemographic characteristics into account. Our results warrant further investigation on the disability incurred by helmintic infections and the usefulness of generic QoL questionnaires in this endeavor.     

A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg⁻¹. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.Tuberculosis, which is caused by Mycobacterium tuberculosis, is a major public health issue worldwide. Because of the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and the high incidence of HIV and tuberculosis coinfection (16), it is becoming increasingly difficult to combat the spread of this disease, and the global health burden of tuberculosis is extremely heavy. The reasons for the persistence of the tubercle bacillus include not only its ability to enter into a state of dormancy in its host for decades, evading the immune system by forming structures called granulomas (17), but also its unique and complex cell wall composed of specific lipids (8). These characteristics are thought to be good focus points for drug development. In granulomas, during the nonreplicative stage, the bacteria have been found to accumulate lipids in the form of intracellular lipid inclusion bodies (LIBs) (13). These lipids are composed mainly of triacylglycerols (TAG) (9, 13) and may originate from the lipolysis of host lipids and/or fatty acid uptake. In fact, M. tuberculosis in the granuloma center can even accumulate lipids originating from the degradation of immune cells (20). In addition, it has been reported that M. tuberculosis internalized by foamy macrophages accumulated LIBs when it joined cell lipid droplets composed of neutral lipids (32). Lipid storage may provide the bacillus with energy via the β-oxidation pathway followed by the glyoxylate cycle, during the chronic phase and the reactivation step (3, 17). These lipids may also supply precursors for the synthesis of bacterial cell membrane lipids, which play a key role in the pathogenicity of M. tuberculosis (4, 23). To investigate the molecular basis of the virulence and pathogenicity of M. tuberculosis, it was therefore proposed to study the lipid metabolism and cell wall remodeling processes in this bacterium.The enzymes involved in the lipid degradation processes induced by this bacterium have attracted considerable attention during the last few years. Based on the complete M. tuberculosis H37Rv genome sequence (6), several open reading frames (ORFs) encoding proteins potentially involved in the lipid metabolism of this strain have been identified, among which are the two lipases from M. tuberculosis that have been purified and characterized so far. Deb et al. identified an enzyme, Rv3097c (LipY), belonging to the hormone-sensitive lipase family, which is able to hydrolyze long-chain TAG (10). A study of LIB mobilization in a lipY-deficient mutant has shown that LipY was involved in TAG hydrolysis under nutriment-deprived conditions (10). LipY may therefore be involved in the degradation of TAG stored during the dormant stage and the subsequent reactivation of the pathogen. In addition, electron microscopy immunolabeling studies of LipY clearly showed that the enzyme had a cell surface localization, thus in direct contact with the host immune system (28). The last identified lipase to date is a monoacylglycerol lipase annotated Rv0183 (7). Like LipY, Rv0183 is located in the cell wall, but its exact physiological function has not yet been elucidated. One hypothesis could be that, like some mammalian cells (e.g., adipocytes), M. tuberculosis expresses several lipolytic enzymes sequentially involved in the lipolysis of TAG (37). The Rv0183 enzyme is conserved in M. bovis (Mb0189) and M. leprae (ML2603), as well as in M. smegmatis (MSMEG_0220), a nonpathogenic mycobacterium which provides a useful model organism and a surrogate host for molecular analysis of M. tuberculosis (19). In order to decipher the cellular role of Rv0183 in M. tuberculosis H37Rv and its contribution to the lipid metabolism of this bacterium, biochemical studies were performed on the homologue MSMEG_0220. For this purpose, the MSMEG_0220 gene from M. smegmatis, encoding a protein showing 68% amino acid sequence identity with Rv0183, was cloned, and the recombinant MSMEG_0220 enzyme (rMSMEG_0220) was produced in Escherichia coli, purified, and biochemically characterized. An M. smegmatis mutant with an MSMEG_0220 disrupted gene was produced to investigate the physiological role of MSMEG_0220.     

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

Apolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.     

Identification and characterization of a brilliant yellow pigment produced by Bordetella pertussis

Culture supernatants of Bordetella pertussis are a brilliant yellow; however, the structure and biological role of the responsible pigment have not been investigated. In this study, a brilliant yellow‐colored fraction was extracted from culture supernatants of B. pertussis and analyzed by HPLC. UV–visible spectral analysis and mass spectrometry identified the brilliant yellow pigment as riboflavin. Riboflavin production was high in lag and early log phases and riboflavin was found to enhance growth of B. pertussis in low‐density cultures. Riboflavin production is not regulated by the BvgAS system. In addition, it was found that other Bordetella species, such as B. parapertussis , B. holmesii and B. bronchiseptica, also release riboflavin into their culture supernatants. This is the first report that B. pertussis secrets riboflavin to the extracellular space and that riboflavin may promote its growth. The mechanism may be associated with pathogenesis of B. pertussis .

     

Genetic Diversity Between Japanese and Chinese Threeline Grunt (<Emphasis Type="Italic">Parapristipoma trilineatum</Emphasis>) Examined by Microsatellite DNA Markers

Threeline grunt (Parapristipoma trilineatum) distributes around the southwestern coast of Japan and the east coast of China. The Chinese P. trilineatum was imported by Japan as an aquacultural seed because of its rapid growth compared with that of the Japanese P. trilineatum. The Japanese P. trilineatum differs from the Chinese P. trilineatum in some quantitative traits, and it has been suggested that these two P. trilineatum populations are genetically different. In order to identify the population structures around Japan and China, 5 local populations of the Japanese P. trilineatum and 2 local populations of the Chinese P. trilineatum were analyzed using 4 microsatellite DNA markers. Significant differences were detected between Japanese and Chinese P. trilineatum and among samples of Chinese P. trilineatum; however, among the samples of Japanese P. trilineatum, no significant differences were detected. These results suggest that care must be taken to prevent the escape of the Chinese P. trilineatum from culture cages around the Japanese coast, in order to preserve the genetically different population structures of Japanese and Chinese P. trilineatum.     

Temporal changes of genetic population structure and diversity in the endangered Blakiston's fish owl (Bubo blakistoni) on Hokkaido Island, Japan, revealed by microsatellite analysis

The Blakiston's fish owl (Bubo blakistoni) population on Hokkaido Island, Japan, decreased to less than one hundred individuals over the last century due to habitat disruption by human activity. Although the ongoing conservation management has slightly restored the population, it remains endangered. In order to assess the genetic variation and population structure of the Blakiston's fish owl in Hokkaido, we genotyped eight microsatellite loci on 120 individuals sampled over the past three decades. The genotype data set showed low levels of genetic variation and gene flow among the geographically isolated five subpopulations. Comparative analysis of past and current populations indicated that some alleles shared by past individuals had been lost, and that genetic variation had declined over the last three decades. The result suggests that the genetic decline may have resulted from inbreeding and/or genetic drift due to bottlenecks in the Hokkaido population. The present study provides invaluable genetic information for the conservation and management of the endangered Blakiston's fish owl in Hokkaido.     

winged eye Induces Transdetermination of Drosophila Imaginal Disc by Acting in Concert with a Histone Methyltransferase,Su(var)3-9

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Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria

Treatment of tuberculosis still represent a major public health issue. The emergence of multi-and extensively-drug resistant (MDR and XDR) Mycobacterium tuberculosis clinical strains further pinpoint the urgent need for new anti-tuberculous drugs. We previously showed that vancomycin can target mycobacteria lacking cell wall integrity, especially those lacking related phthiocerol and phthiodolone dimycocerosates, PDIM A and PDIM B, respectively. As aloe emodin was previously hypothesized to be able to target the synthesis of mycobacterial cell wall lipids, we tested its ability to potentiate glycopeptides antimycobacterial activity. The aloe emodin with the vancomycin induced a combination effect beyond simple addition, close to synergism, at a concentration lower to reported IC₅₀ cytotoxic value, on M. bovis BCG and on H37Rv M. tuberculosis. Interestingly, out of six MDR and pre-XDR clinical strains, one showed a strong synergic susceptibility to the drug combination. Mycobacterial cell wall lipid analyses highlighted a selective reduction of PDIM B by aloe emodin.