Phase I study of intradermal immunotherapy with oil-attached Mycobacterium smegmatis cell wall skeleton and trehalose dimycolate

Summary The clinical toxicity of intradermal immunotherapy with a nonviable mycobacterial vaccine consisting of oil-attached Mycobacterium smegmatis cell wall skeleton (CWS) and trehalose dimycolate (P3) was evaluated. Fifteen patients with advanced hypernephroma, lung cancer, or malignant melanoma were evaluated. Patients received one to ten separate intradermal injections in the subclavicular areas weekly for up to 8 weeks. Each separate injection usually contained 75 g CWS and 37.5 g P3.There were few systemic side effects. Mild fever occurred in 30% of 69 treatments. Severe local toxicity with ulceration and/or abscess formation occurred in seven patients. Regression of disease was observed in one patient to occur on two separate occasions following separate courses of therapy.Although intradermal CWS/P3 can be locally toxic, treatment with up to four separate injections of 75 g CWS combined with 37.5 g P3 every 1–2 weeks appears appropriate, from this study, for additional clinical trials.     

Mannosyltransferase is required for cell wall biosynthesis, morphology and control of asexual development in Neurospora crassa

Two Neurospora mutants with a phenotype that includes a tight colonial growth pattern, an inability to form conidia and an inability to form protoperithecia have been isolated and characterized. The relevant mutations were mapped to the same locus on the sequenced Neurospora genome. The mutations responsible for the mutant phenotype then were identified by examining likely candidate genes from the mutant genomes at the mapped locus with PCR amplification and a sequencing assay. The results demonstrate that a map and sequence strategy is a feasible way to identify mutant genes in Neurospora. The gene responsible for the phenotype is a putative alpha-1,2-mannosyltransferase gene. The mutant cell wall has an altered composition demonstrating that the gene functions in cell wall biosynthesis. The results demonstrate that the mnt-1 gene is required for normal cell wall biosynthesis, morphology and for the regulation of asexual development.     

Trehalose is required for growth of Mycobacterium smegmatis

Mycobacteria contain high levels of the disaccharide trehalose in free form as well as within various immunologically relevant glycolipids such as cord factor and sulfolipid-1. By contrast, most bacteria use trehalose solely as a general osmoprotectant or thermoprotectant. Mycobacterium tuberculosis and Mycobacterium smegmatis possess three pathways for the synthesis of trehalose. Most bacteria possess only one trehalose biosynthesis pathway and do not elaborate the disaccharide into more complex metabolites, suggesting a distinct role for trehalose in mycobacteria. We disabled key enzymes required for each of the three pathways in M. smegmatis by allelic replacement. The resulting trehalose biosynthesis mutant was unable to proliferate and enter stationary phase unless supplemented with trehalose. At elevated temperatures, however, the mutant was unable to proliferate even in the presence of trehalose. Genetic complementation experiments showed that each of the three pathways was able to recover the mutant in the absence of trehalose, even at elevated temperatures. From a panel of trehalose analogs, only those with the native alpha,alpha-(1,1) anomeric stereochemistry rescued the mutant, whereas alternate stereoisomers and general osmo- and thermoprotectants were inactive. These findings suggest a dual role for trehalose as both a thermoprotectant and a precursor of critical cell wall metabolites.     

Permeability of the cell wall of Mycobacterium smegmatis

The cell wail of Mycobacterium smegmatis me²155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to β-lactams ranged from 10 × 10 ⁻⁷ to 0.5 × 10 ⁻⁷ cm s⁻¹. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel-forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage-gated cation-selective channel. The mycobacterial porin channel appeared to be water-filled since the single-channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single-channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point-charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10- to 20-fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.     

Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids

A mutant strain of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids was recently isolated (Liu, J., and Nikaido, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4011-4016). This mutant failed to synthesize full-length mycolic acids and accumulated a series of long chain beta-hydroxymeromycolates. In this work, we provide a detailed characterization of the localization of meromycolates and of the cell wall structure of the mutant. Thin layer chromatography showed that the insoluble cell wall matrix remaining after extraction with chloroform/methanol and SDS still contained a large portion of the total meromycolates. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectroscopy analysis of fragments arising from Smith degradation of the insoluble cell wall matrix revealed that the meromycolates were covalently attached to arabinogalactan at the 5-OH positions of the terminal arabinofuranosyl residues. The arabinogalactan appeared to be normal in the mutant strain, as analyzed by NMR. Analysis of organic phase lipids showed that the mutant cell wall contained some of the extractable lipids but lacked glycopeptidolipids and lipooligosaccharides. Differential scanning calorimetry of the mutant cell wall failed to show the large cooperative thermal transitions typical of intact mycobacterial cell walls. Transmission electron microscopy showed that the mutant cell wall had an abnormal ultrastructure (without the electron-transparent zone associated with the asymmetric mycolate lipid layer). Taken together, these results demonstrate the importance of mycolic acids for the structural and functional integrity of the mycobacterial cell wall. The lack of highly organized lipid domains in the mutant cell wall explains the drug-sensitive and temperature-sensitive phenotypes of the mutant.     

A tetrameric porin limits the cell wall permeability of Mycobacterium smegmatis

Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant against many antibiotics. Pore proteins mediate the diffusion of hydrophilic nutrients across this membrane. Electron microscopy revealed that the outer membrane of Mycobacterium smegmatis contained about 1000 protein pores per microm(2), which are about 50-fold fewer pores per microm(2) than in Gram-negative bacteria. The projection structure of the major porin MspA of M. smegmatis was determined at 17 A resolution. MspA forms a cone-like tetrameric complex of 10 nm in length with a single central pore. Thus, MspA is drastically different from the trimeric porins of Gram-negative bacteria and represents a new class of channel proteins. The formation of MspA micelles indicated that the ends of MspA have different hydrophobicities. Oriented insertion of MspA into membranes was demonstrated in lipid bilayer experiments, which revealed a strongly asymmetrical voltage gating of MspA channels at -30 mV. The length of MspA is sufficient to span the outer membrane and contributes in combination with the tapering end of the pore and the low number of pores to the low permeability of the cell wall of M. smegmatis for hydrophilic compounds.     

A lipoprotein modulates activity of the MtrAB two‐component system to provide intrinsic multidrug resistance,cytokinetic control and cell wall homeostasis in Mycobacterium

The MtrAB signal transduction system, which participates in multiple cellular processes related to growth and cell wall homeostasis, is the only two‐component system known to be essential in Mycobacterium. In a screen for antibiotic resistance determinants in Mycobacterium smegmatis, we identified a multidrug‐sensitive mutant with a transposon insertion in lpqB, the gene located immediately downstream of mtrA–mtrB. The lpqB mutant exhibited increased cell–cell aggregation and severe defects in surface motility and biofilm growth. lpqB cells displayed hyphal growth and polyploidism, reminiscent of the morphology of Streptomyces, a related group of filamentous Actinobacteria. Heterologous expression of M. tuberculosis LpqB restored wild‐type characteristics to the lpqB mutant. LpqB interacts with the extracellular domain of MtrB, and influences MtrA phosphorylation and promoter activity of dnaA, an MtrA‐regulated gene that affects cell division. Furthermore, in trans expression of the non‐phosphorylated, inactive form of MtrA in wild‐type M. smegmatis resulted in phenotypes similar to those of lpqB deletion, whereas expression of the constitutively active form of MtrA restored wild‐type characteristics to the lpqB mutant. These results support a model in which LpqB, MtrB and MtrA form a three‐component system that co‐ordinates cytokinetic and cell wall homeostatic processes.     

MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the DeltamspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the DeltamspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the DeltamspA mutant [corrected] This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.     

Trehalose 6‐phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigatus

The trehalose biosynthesis pathway is critical for virulence in human and plant fungal pathogens. In this study, we tested the hypothesis that trehalose 6‐phosphate phosphatase (T6PP) is required for Aspergillus fumigatus virulence. A mutant of the A. fumigatus T6PP, OrlA, displayed severe morphological defects related to asexual reproduction when grown on glucose (1%) minimal media. These defects could be rescued by addition of osmotic stabilizers, reduction in incubation temperature or increase in glucose levels (> 4%). Subsequent examination of the mutant with cell wall perturbing agents revealed a link between cell wall biosynthesis and trehalose 6‐phosphate (T6P) levels. As expected, high levels of T6P accumulated in the absence of OrlA resulting in depletion of free inorganic phosphate and inhibition of hexokinase activity. Surprisingly, trehalose production persisted in the absence of OrlA. Further analyses revealed that A. fumigatus contains two trehalose phosphorylases that may be responsible for trehalose production in the absence of OrlA. Despite a normal growth rate under in vitro growth conditions, the orlA mutant was virtually avirulent in two distinct murine models of invasive pulmonary aspergillosis. Our results suggest that further study of this pathway will lead to new insights into regulation of fungal cell wall biosynthesis and virulence.     

Effects of polyanions and polycations on the trehalose phosphate synthetase of Mycobacterium smegmatis

When tested as activators on the trehalose phosphate synthetase [UDP-d-glucose:d-glucose 6-phosphate α-d-glucosyltransferase, EC 2.4.1.15 (46)] from Mycobacterium smegmatis, heparin was the best, various other sulfated polysaccharides (especially chondroitin 4- and 6-sulfates, dermatan sulfate, heparan sulfate, and γ-carrageenan) and polynucleotides were good, but hyaluronic acid, d-galacturonan, dextran sulfate, and keratan sulfate, were poor. Digestion of chondroitin sulfate with hyaluronidase destroyed the activating ability, but separation of the digestion products on Sephadex G-100 resin gave large-molecular-weight componentns that still showed activating ability. A sulfated tetra- or octa-saccharide isolated from chondroitin sulfate did not activate the enzyme, nor did they prevent the activation by chondroitin sulfate, suggesting that these small polyanions do not bind to the enzyme. Among polycations, poly-dl-ornithine (mol. wt. 15,600 daltons) was the best inhibitor of the enzyme followed by poly-l-lysine (mol. wt. 4,000 daltons), poly-d-lysine (mol. wt. 70,000 daltons), poly-d,l-lysine (mol. wt. 35,000 daltons), and then poly-l-ornithine (mol. wt. 120,000 daltons); polyglycine, polyleucine, and polyhistidine showed no effect. In all cases, more polycation was required to inhibit the enzyme when heparin was used as the activator than when chondroitin sulfate was used. The order of mixing of various reaction components was important for the extent of inhibition, the greates inhibition being observed when polyanion and polycation were mixed before the addition of enzyme, and the smallest when polyanion and enzyme were mixed before the addition of polycation.     

Binding sites of sorbed uranyl ion in the cell wall of Mycobacterium smegmatis

Abstract A study of cell-wall site interaction of uranyl ion adsorbed by non-proliferative suspensions of Mycobacterium smegmatis at pH 1 has been carried out using extracts of arabinogalactan-peptidoglycan and phospholipids obtained from whole cells treated under sorption conditions. Evidence for binding of UO₂²⁺ by constituent P-lipids was provided by comparative ³¹P-NMR and IR spectroscopic measurements of the isolated wall fractions and of the model complex uranyl-phosphatidyl inositol.     

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

A number of pharmaceutical steroid synthons are currently produced through the microbial side‐chain cleavage of natural sterols as an alternative to multi‐step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc²155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc²155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (KshAB) and a ketosteroid Δ¹‐dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4‐androstadiene‐3,17‐dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039‐5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4‐androstene‐3,17‐dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc²155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.     

The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of DeltatatA and DeltatatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active beta-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active 'BlaC. Thus, 'BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.