Surface-exposed glycopeptidolipids of Mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids

Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.     

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.     

Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild-type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O-methylated rhamnose and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild-type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.     

Identification and characterization of novel immunogenic proteins of Streptococcus suis serotype 2

Streptococcus suis, a zoonotic pathogen, caused serious outbreaks in humans with high mortality rates in the past decade. To develop safer and more effective vaccines, particularly for human protection, cell wall and extracellular proteins of S. suis serotype 2 were analyzed by an immunoproteomic approach in this study. Thirty-two proteins with high immunogenicity were identified and 22 of them were newly identified. Further analyses of 9 selected proteins revealed that (1) these 9 proteins were expressed in all tested virulent S. suis serotype 2 isolates, (2) antisera against 6 of the selected proteins efficiently killed the bacteria by opsonized phagocytosis in human blood, and (3) significantly higher levels of serum antibodies against 3 proteins were detected in both patients and infected swines. Therefore, our results suggest the 3 proteins (SSU98_0197, SSU98_1094 and SSU1664) have strong potential to be vaccine candidates.     

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare

Abstract Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M. intracellulare that is absent in M. avium . This antigen cross-reacts immunologically with a major 38 kDa antigen of M. tuberculosis , and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli . Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.     

Identification of antibody responses to the serotype-nonspecific molecular species of glycopeptidolipids in Mycobacterium avium infection

Glycopeptidolipids (GPLs) comprise a major surface glycolipid of Mycobacterium avium complex (MAC), and their unique oligosaccharide extensions are known to define MAC serotypes. Beside the mature form of “serotype-specific” GPLs (ssGPLs), those that share the backbone structure but lack the oligosaccharide extensions exist as abundantly in all MAC serotypes, but the presumption was that antibody responses might not be directed to these “serotype-nonspecific” GPLs (nsGPLs) due to the lack of the sugar chain epitope. Here, we show that IgG responses to nsGPLs indeed occur in MAC-infected guinea pigs. The pool of anti-nsGPL antibodies was distinct from that of anti-ssGPL antibodies in terms of requirements for the oligosaccharide and acetylation for their target recognition. Because nsGPLs are shared in virtually all MAC strains, but totally absent in Mycobacterium tuberculosis, this study suggests that detecting serum anti-nsGPL antibodies can potentially be useful for differential diagnosis of MAC infection and tuberculosis.     

Mycobacterium avium 104 deleted of the methyltransferase D gene by allelic replacement lacks serotype-specific glycopeptidolipids and shows attenuated virulence in mice

Mycobacterium avium is a major opportunistic pathogen of AIDS patients in the United States. The understanding of M. avium pathogenesis has been hampered by the inability to create gene knockouts by homologous recombination, an important mechanism for defining and characterizing virulence factors. In this study a functional methyltransferase D (mtfD) gene was deleted by allelic replacement in the M. avium strain 104. Methyltransferase D is involved in the methylation of glycopeptidolipids (GPLs); highly antigenic glycolipids found in copious amounts on the M. avium cell surface. Interestingly, the loss of mtfD resulted in M. avium 104 containing only the non-serotype specific GPL. Results also suggest that the mtfD encodes for a 3-O-methyltransferase. The absence of significant amounts of any serotype-specific GPLs as a consequence of mtfD deletion indicates that the synthesis of the core 3,4-di-O-methyl rhamnose is a prerequisite for synthesis of the serotype-specific GPLs. Macrophages infected with the mtfD mutant show elevated production of tumour necrosis factor-alpha (TNF-alpha) and RANTES compared to control infections. In addition, the M. avium 104 mtfD mutant exhibits decreased ability to survive/proliferate in mouse liver and lung compared to wild-type 104, as assessed by bacterial counts. Importantly, the mtfD mutant complemented with a wild-type mtfD gene maintained an infection level similar to wild-type. These experiments demonstrate that the loss of mtfD results in a M. avium 104 strain, which preferentially activates macrophages in vitro and shows attenuated virulence in mice. Together our data support a role for GPLs in M. avium pathogenesis.     

Identification and characterization of two adenosine phosphorylase activities in Mycobacterium smegmatis

Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-d-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K_m (K_{m app}) of 98 ± 6 μM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5′-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.     

Nucleotide sequence analysis and serologic characterization of the Mycobacterium intracellulare homologue of the Mycobacterium tuberculosis 19 kDa antigen

Disseminated Mycobacterium avium/Mycobacterium intracellulare complex (MAC) disease is a frequent complication in patients with the acquired immune deficiency syndrome (AIDS). In this report, we present the nucleotide sequence of the M. intracellulare MI22 gene. Computer sequence comparisons reveal that the MI22 gene, which encodes a serologically active protein, has 78% DNA sequence identity and 77% protein sequence identity with the seroreactive 19 kDa Mycobacterium tuberculosis lipoprotein antigen. Southern blot hybridizations indicate that an MI22 gene probe binds similar-sized restriction fragments in M. tuberculosis and M. intracellular genomic DNA. In addition, immunoblot analyses demonstrate that MI22 is recognized by sera from tuberculosis patients. These data further support the existence of 19 kDa MAC and M. tuberculosis protein homologues. Phase partitioning experiments and the presence of a consensus lipid modification site in the deduced MI22 protein sequence strongly suggest that M122 is also a lipoprotein. Comparative analyses of these mycobacterial antigenic homologues may provide the basis for the design of species-specific diagnostic reagents.     

Identification and characterization of two flavohemoglobin genes in Dictyostelium discoideum

Flavohemoglobins are being identified in an expanding number of prokaryotes and unicellular eukaryotes. These molecules consist of an N-terminal hemoglobin domain and a C-terminal oxidoreductase domain, and are considered to function in storage or as sensors for O2, and in defense against oxidative stress and/or NO. However, their physiological significance has not yet been determined. Here, we isolated and analyzed two flavohemoglobin genes of Dictyostelium discoideum, DdFHa and DdFHb, which lie close to each other in the genome. DdFHs were induced by submerged conditions, and enriched in the sexually mature cells of D. discoideum. Although they were not essential for growth or development under standard laboratory conditions, disruption of both genes caused an increase in number of large but uninuclear cells, and hypersensitivity to higher concentrations of glucose and to NO releasers. These results indicate that DdFHs are responsible for transducing NO signals to maintain normal cellular conditions against environmental stresses.     

Identification of two novel primate-specific genes in DSCR.

We recently helped to complete the sequence of human chromosome 21 at a very high level of accuracy. Using this sequence we identified two novel genes, designated DSCR9 and DSCR10, in the so-called Down Syndrome Critical Region (DSCR) by computational gene prediction and subsequent cDNA cloning. Both DSCR9 and DSCR10 are expressed preferentially in testis and encode functionally unknown proteins with 149 and 87 amino acid residues, respectively. Zoo blot analysis suggested that both genes are exclusive to primate genomes such as chimpanzee, gorilla, orangutan, crab-eating monkey and African green monkey but are not present in other non-primate mammals including mouse, dog, cat, and chicken. Comparative genomic sequence analysis of DSCR9 and DSCR10 with the corresponding mouse syntenic region confirmed the lack of these genes in the mouse. These results strongly suggest that DSCR9 and DSCR10 have emerged as a new class of gene in the primate lineage during evolution.     

Identification and characterization of two novel low-molecular-weight dual specificity phosphatases

We have cloned and characterized two novel human low molecular weight dual specificity phosphatases (LMW-DSPs). Both genes are expressed exclusively in the testis, but are not altered in any of several disease states examined. Transfection into COS cells indicates that both proteins are expressed in the nucleus and the cytoplasm. Both proteins are able to dephosphorylate the phosphotyrosine analog pNPP in vitro and can be inhibited by sodium orthovanadate. In vitro experiments also demonstrate that both DSPs can dephosphorylate single and diphosphorylated synthetic MAPK peptides, with preference for the phosphotyrosine and diphosphorylated forms over phosphothreonine. However, when co-transfected with MAPKs into COS cells, the novel DSPs exhibited no detectable in vivo activity against MAPKs under our conditions. Our data suggest that these novel LMW-DSPs might belong to a new subclass of testis-specific proteins that act independently of the MAPK signal transduction cascade and do not depend on N-terminal docking regions for substrate binding.     

Identification and characterization of a novel protein that regulates RNA-protein interaction

In a previous study [Nachaliel et al., 1993], we identified an RNA-binding protein (RBP) in FTO-2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor. We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA-binding subunit(s) (RBS), and regulatory subunit(s) (RS). We purified the RS to near-homogeneity (Mr approximately 25,000) and determined the amino acid sequence of a peptide derived from RS. On the basis of this sequence information, the cDNA for RS was obtained. Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA-binding activity. The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS-polyacrylamide gels. Sequence comparison showed that RS is almost identical to DJ-1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein. However, the protein does not contain any known motifs that can provide a clue as to its exact function. Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus. The possible role of RS is discussed.