Bioconversion of Phytosterols into Androstenedione by Mycobacterium.

The chapter describes the bioconversion of phytosterols to androstenedione (AD) with Mycobacterium spp. in shake flasks and fermenters, as well as LC-MS based methods for analysis of phytosterols and steroid products.Phytosterols are derived as a by-product of vegetable oil refining and of manufacture of wood pulp. Phytosterols contain the same four-ring nucleus as steroids, and may be converted to high-value steroids by removing the side chain at C17 and minor changes at other sites in the ring structure.Many bacteria, including Mycobacterium spp., are able to degrade phytosterols. Mutants of Mycobacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and/or androstadienedione (ADD).The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic to cells.Two main strategies have been employed to overcome this challenge: the use of two-phase systems, and the addition of chemically modified cyclodextrins. The latter method is used here.Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as suggestions to minimize the practical problems caused by the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.

Role Identification and Application of SigD in the Transformation of Soybean Phytosterol to 9α-Hydroxy-4-androstene-3,17-dione in Mycobacterium neoaurum.

9α-Hydroxy-4-androstene-3,17-dione (9-OHAD) is a valuable steroid pharmaceutical intermediate which can be produced by the conversion of soybean phytosterols in mycobacteria. However, the unsatisfactory productivity and conversion efficiency of engineered mycobacterial strains hinder their industrial applications. Here, a sigma factor D (sigD) was investigated due to its dramatic downregulation during the conversion of phytosterols to 9-OHAD. It was determined as a negative regulator in the metabolism of phytosterols, and the deletion of sigD in a 9-OHAD-producing strain significantly enhanced the titer of 9-OHAD by 18.9%. Furthermore, a high yielding strain was constructed by the combined modifications of sigD and choM2, a key gene in the phytosterol metabolism pathway. After the modifications, the productivity of 9-OHAD reached 0.071 g/L/h (10.27 g/L from 20 g/L phytosterol), which was 22.5% higher than the original productivity of 0.058 g/L/h (8.37 g/L from 20 g/L phytosterol) in the industrial resting cell biotransformation system.

Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome.

Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1ß/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1ß processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1ß expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.

Folate-targeted hapten immunotherapy of adjuvant-induced arthritis: comparison of hapten potencies.

We have previously reported that disease symptoms can be greatly ameliorated in rodents with adjuvant-induced arthritis (AIA) by first immunizing the rodents against fluorescein and then treating the animals with folate-fluorescein. In this targeted hapten therapy, folate-fluorescein was shown to decorate folate receptor (FR)-expressing activated macrophages with fluorescein (an immunogenic hapten), leading to binding of antifluorescein antibodies and the consequent elimination of the activated macrophages by Fc receptor-expressing immune cells. In the current study, we compare the therapeutic potencies of a variety of FR-targeted haptens in treating the symptoms of AIA in rats. Rats were immunized with either dinitrophenyl (DNP) or trinitrophenyl (TNP) conjugated to keyhole limpet hemocyanin followed by induction of AIA with heat-inactivated Mycobacterium butyricum. Following development of arthritis, rats were treated with one of five folate-hapten conjugates (folate-DNP1, folate-DNP2, folate-DNP3, folate-FITC, or folate-TNP) at two different doses (30 nmol/kg or 200 nmol/kg) 5x/week for 25 days. Symptoms of AIA in treated rats, including paw swelling, arthritis score, splenomegaly, bone erosion, and FR(+) activated macrophage density in inflamed tissues, were quantitated over the course of therapy. Although all folate-hapten conjugates promoted a reduction in disease symptoms, folate-TNP and folate-FITC proved to be more potent than any of the 3 folate-DNP conjugates. We conclude that both folate-TNP and folate-FITC constitute promising haptens for use in FR-targeted immunotherapy of arthritis.

Phosphatidylinositol mannosides are efficient mucosal adjuvants.

The development of defined sub-unit vaccines requires the inclusion in the vaccine of an immunological adjuvant. The most important property of adjuvants for vaccines aimed at inducing optimal protection against intracellular bacteria such as Mycobacterium tuberculosis or M. bovis is the ability to enhance cell-mediated immunity, specifically Th1 responses. In this paper, we describe a system where transgenic mice expressing a high proportion of T cells specific for an ovalbumin (OVA) peptide are used to assess the ability of a novel class of adjuvants to positively modulate cell-mediated immune responses. Defined fractions containing purified native or synthetic phosphatidylinositol mannosides (PIMs) from mycobacteria were assessed for their adjuvant activities in response to the model antigen (OVA). Purified PIM preparations given to mice with OVA by the subcutaneous route were shown to elicit an enhanced release of interferon-gamma (IFN-gamma) in cellular responses to OVA peptide in vitro. Very little interleukin-4 (IL-4) was released by cells from mice immunized with PIMs and OVA, whereas cells from animals immunized with complete Freund's adjuvant (CFA) and OVA released IL-4 as well as IFN-gamma. Synthetic preparations of PIM2 and PIM4 also acted as adjuvants in the mouse model studied. In addition, PIM preparations were shown to generate an efficient cell-mediated immune response to OVA, when the antigen/adjuvant preparations were administered via the oral route or intranasal route. PIM preparations elicited substantial release of interleukin-12 (IL-12) from dendritic cells (DCs). These data suggest that purified or synthetic PIMs act as adjuvants when administered at mucosal surfaces and represent a new class of adjuvants for mucosal immunization against intracellular pathogens.

Altered expression profile of mycobacterial surface glycopeptidolipids following treatment with the antifungal azole inhibitors econazole and clotrimazole.

The azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium avium-intracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis, produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis. In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis, suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N-acyl chain in GPL biosynthesis. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum, M. peregrinum, M. chelonae and M. abscessus.

Mycobacterium cosmeticum sp. nov., a novel rapidly growing species isolated from a cosmetic infection and from a nail salon.

Four isolates of a rapidly growing Mycobacterium species had a mycolic acid pattern similar to that of Mycobacterium smegmatis, as determined by HPLC analyses. Three of the isolates were from footbath drains and a sink at a nail salon located in Atlanta, GA, USA; the fourth was obtained from a granulomatous subdermal lesion of a female patient in Venezuela who was undergoing mesotherapy. By random amplified polymorphic DNA electrophoresis and PFGE of large restriction fragments, the three isolates from the nail salon were shown to be the same strain but different from the strain from the patient in Venezuela. Polymorphisms in regions of the rpoB, hsp65 and 16S rRNA genes that were shown to be useful for species identification matched for the two strains but were different from those of other Mycobacterium species. The 16S rRNA gene sequence placed the strains in a taxonomic group along with Mycobacterium frederiksbergense, Mycobacterium hodleri, Mycobacterium diernhoferi and Mycobacterium neoaurum. The strains produced a pale-yellow pigment when grown in the dark at the optimal temperature of 35 degrees C. Biochemical testing showed that the strains were positive for iron uptake, nitrate reduction and utilization of d-mannitol, d-xylose, iso-myo-inositol, l-arabinose, citrate and d-trehalose. The strains were negative for d-sorbitol utilization and production of niacin and 3-day arylsulfatase, although arylsulfatase activity was observed after 14 days. The isolates grew on MacConkey agar without crystal violet but not on media containing 5 % (w/v) NaCl or at 45 degrees C. They were susceptible to ciprofloxacin, amikacin, tobramycin, cefoxitin, clarithromycin, doxycycline, sulfamethoxazole and imipenem. The name Mycobacterium cosmeticum sp. nov. is proposed for this novel species; two strains, LTA-388(T) (=ATCC BAA-878(T)=CIP 108170(T)) (the type strain) and 2003-11-06 (=ATCC BAA-879=CIP 108169) have been designated, respectively, for the strains of the patient in Venezuela and from the nail salon in Atlanta, GA, USA.

Characterization of multiple-substrate utilization by anthracene-degrading Mycobacterium frederiksbergense LB501T.

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by Mycobacterium frederiksbergense LB501T under various substrate regimens. The distinct (13)C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that M. frederiksbergense LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of M. frederiksbergense LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by M. frederiksbergense LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.

Drug-disease interactions: reduced beta-adrenergic and potassium channel antagonist activities of sotalol in the presence of acute and chronic inflammatory conditions in the rat.

Inflammation may influence response to pharmacotherapy. We investigated the effect of inflammation on response to sotalol, a beta-adrenergic receptor and potassium channel antagonist. Racemic sotalol (40 mg kg(-1)) was administered to healthy, acutely (interferonalpha 2a-induced) and chronically (Mycobacterium butyricum-induced adjuvant arthritis) inflamed male Sprague-Dawley rats (n=4 - 6/group). Another group of interferon-treated rats received 3 mg kg(-1) of anti-TNF antibody infliximab. Electrocardiogram (ECG) recorded and plasma sotalol concentration monitored for 6 h. The study was repeated in acutely inflamed rats following administration of stereochemically pure individual sotalol enantiomers [40 mg kg(-1) S (potassium channel blocker) or 20 mg kg(-1) R (beta-adrenergic/potassium channel blocker)]. Chronic arthritis was readily evident. Acute arthritis was associated with elevated segmented neutrophils and increased plasma nitrite and tumour necrosis factor (TNF) concentrations. Sotalol affected ECG in all rats. In both inflamed groups, however, response to sotalol in prolongation of QT interval (potassium channel sensitivity) was reduced. The effect of PR interval (beta-adrenergic activity) was also reduced following administration of the racemate and R-enantiomer. No significant differences in pharmacokinetics were observed between control and inflamed rats. Infliximab reduced nitrite and TNF concentrations and reversed the effect of acute inflammation on both PR and QT intervals. The reduced electrocardiographic responses to sotalol is likely due to the influence of inflammation on the action of the drug on both beta-adrenergic and potassium channel receptors secondary to over-expression of pro-inflammatory cytokines and/or nitric oxide. Our observation may have therapeutic consequences in all conditions where inflammatory mediators are increased.

Use of gas chromatographic fatty acid and mycolic acid cleavage product determination to differentiate among Mycobacterium genavense, Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium tuberculosis.

Three Mycobacterium genavense strains and three American Type Culture Collection reference strains each of Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium tuberculosis were subcultured onto Mycobacteria 7H11 agar (Difco Laboratories, Detroit, Mich.) supplemented with mycobactin J (Allied Laboratories, Fayette, Mo.). After 4 weeks of incubation at 37 degrees C in 10% CO2, the cultures were analyzed by gas-liquid chromatography (GLC) for their fatty acids and mycolic acid cleavage products. M. fortuitum was clearly differentiated from M. genavense by the presence of the specific marker 2-methyloctadecenoic acid in M. fortuitum and by the ratio of tetracosanoic acid to hexacosanoic acid. This ratio was <1 for M. genavense and >3 for M. fortuitum. M. fortuitum also contained docosanoic acid, which was not detected in M. genavense. M. genavense, M. simiae, and M. tuberculosis, which have similar GLC profiles, were also differentiated from each other by the presence of either cis-10-hexadecenoic acid or cis-11-hexadecenoic acid and by tetradecanoic acid content.

[Study on the enhancing effect of polyporus polysaccharide, mycobacterium polysaccharide and lentinan on lymphokine-activated killer cell activity in vitro].

The actions of Polyporus polysaccharide (PPS), mycobacterium polysaccharide (MPS) and lentinan (LEN) on lymphokine-activated killer (LAK) cell activity in vitro were investigated in this study. Human peripheral blood mononuclear cells (PBMC) were cultured for 96 hours with medium containing different concentrations of the above-mentioned drugs in combination with recombinant interleukin 2 (rIL-2). Then cell-mediated lysis was determined by 1H-TdR release assay including NK sensitive and NK resistant target cells. The results demonstrated that, when combined with rIL-2 in a certain concentration, all three kinds of polysaccharides could enhance the LAK activity by 42%-56.9%, and reduce the dose of rIL-2 by 50% (P < 0.05-0.01). It suggested that the PPS, MPS and LEN could be used as bioactivity regulators in LAK cell therapy in tumor treatment.