Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis

Brzostek, Anna; Śliwiński, Tomasz; Rumijowska-Galewicz, Anna; Korycka-Machała, Małgorzata; Dziadek, Jarosław

doi:10.1099/mic.0.27953-0

Cited by 54 publications

(64 citation statements)

References 26 publications

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“…The mechanistic determinants for the KshAB low turnover number for 1,4-BNC compared with other ⌬ 1,4 substrates are not clear from the available data. The relative substrate specificities of KshAB for ⌬ 4 and ⌬ 1,4 compounds appear to differ between species, with KshAB from Mycobacterium smegmatis exhibiting a much lower capacity to transform ⌬ 4 steroids than Mtb KshAB, based on metabolite accumulation studies in ⌬kstD knock-out cultures (18,19).…”

Section: Discussionmentioning

confidence: 99%

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis

Capyk

Casabon

Gruninger

et al. 2011

Journal of Biological Chemistry

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Background: Mycobacterium tuberculosis (Mtb) degrades cholesterol throughout its infection cycle. Results: Cholesterol ring-degrading enzymes have higher activities with side chain degradation intermediates than with compounds with fully degraded side chains. Conclusion: Cholesterol side chain and ring degradation occur concurrently. Significance: Understanding bacterial cholesterol catabolism facilitates the design of novel therapeutics and the production of high value steroids.

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Section: Discussionmentioning

confidence: 99%

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis

Capyk

Casabon

Gruninger

et al. 2011

Journal of Biological Chemistry

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“…The bacterial cells were spun down, washed five times for removal of extracellular cholesterol, and extracted three times with an equal volume of chloroform. To quantify the accumulation of cholesterol, equal amounts of 4-androstene-3,11,17-trione (Sigma) were added to each sample as an internal standard, and samples were subjected to gas chromatography as previously described (5). To obtain samples of the M. tuberculosis cell wall free-lipid zone and defatted cells, pellets were obtained from 20 ml of M. tuberculosis culture, washed five times, and extracted three times with an equal volume of chloroform-methanol (2:1, vol/vol) for 48 h at room temperature on a rotatory shaker (200 rpm).…”

Section: Methodsmentioning

confidence: 99%

“…It is well known that fast-growing mycobacteria degrade natural sterols and use them as a source of carbon and energy (5,20,22). However, the ability of tubercle bacilli to utilize cholesterol VOL.…”

Section: Mmentioning

confidence: 99%

“…It is well known that cholesterol can be utilized by fastgrowing, nonpathogenic mycobacteria (5,20,22), but it was previously thought that pathogenic mycobacteria might not be able to use cholesterol as a carbon and energy source (3). Recently, however, bioinformatic analysis identified a cassette of cholesterol catabolism genes in actinomycetes, including the M. tuberculosis complex (41).…”

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confidence: 99%

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Mycobacterium tuberculosis Is Able To Accumulate and Utilize Cholesterol

et al. 2009

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It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.Mycobacterium tuberculosis, the causative agent of tuberculosis, is a very successful pathogen that infects one-third of the human population (21). Only 10% of primary infected individuals develop active disease during their lifetimes. Tubercle bacilli are able to persist in a dormant state, from which they may reactivate and induce the contagious disease state (13). In asymptomatic hosts, M. tuberculosis exists in reservoirs called granulomas, which are cellular aggregates that restrict bacterial spreading (40). Granulomas are organized collections of mature macrophages that exhibit a certain typical morphology and that arise in response to persistent intracellular pathogens (1, 4). Pathogenic mycobacteria can induce the formation of foamy macrophages filled with lipid-containing bodies; these have been postulated to act as a secure, nutrient-rich reservoir for tubercle bacilli (31). Moreover, M. tuberculosis DNA has been detected in fatty tissues surrounding the kidneys, as well as those of the stomach, lymph nodes, heart, and skin. Tubercle bacilli are able to enter adipocytes, where they accumulate within intracytoplasmic lipid inclusions and survive in a nonreplicating state (26). In vivo, it is expected that M. tuberculosis adapts metabolically to nutrient-poor conditions characterized by glucose deficiency and an abundance of fatty acids (25,26). The presence of a complex repertoire of lipid metabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important alternative carbon and energy sources for this pathogen (7).One attractive potential alternative nutrient that is readily available in the mammalian host is cholesterol, a major sterol of the plasma membrane. The presence of cholesterol in lipid rafts is required in order for microorga...

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“…1) (Szentirmai, 1990). If the enzyme activity of KSDD is inactivated or reduced, the molar ratio of AD/ADD in the production mixture will increase (Choi et al, 1995;Brzostek et al, 2005;Wei et al, 2010b). Previously, our laboratory had isolated the ZAD strain of M. neoaurum (Fig.…”

Section: Introductionmentioning

confidence: 99%

Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment

Liu

Zhang

Rao

et al. 2015

J. Zhejiang Univ. Sci. B

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Steroid medication is used extensively in clinical applications and comprises a large and vital part of the pharmaceutical industry. However, the difficulty of separating 4-androstene-3,17-dione (AD) from 1,4-androstadiene-3,17-dione (ADD) restricts the application of the microbial transformation of phytosterols in the industry. A novel atmospheric and room temperature plasma (ARTP) treatment, which employs helium as the working gas, was used to generate Mycobacterium neoaurum mutants producing large amounts of AD. After treatment of cultures with ARTP, four mutants were selected using a novel screening method with a color assay. Among the mutants, M. neoaurum ZADF-4 was considered the best candidate for industrial application. When the fermentation medium contained 15 g/L phytosterols and was cultivated on a rotary shaker at 160 r/min at 30 °C for 7 d, (6.28±0.11) g/L of AD and (0.82±0.05) g/L of ADD were produced by the ZADF-4 mutant, compared with (4.83±0.13) g/L of AD and (2.34±0.06) g/L of ADD by the original strain, M. neoaurum ZAD. Compared with ZAD, the molar yield of AD increased from 48.3% to 60.3% in the ZADF-4 mutant. This result indicates that ZADF-4 may have potential for industrial production of AD.

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Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis

Cited by 54 publications

References 26 publications

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis

Mycobacterium tuberculosis Is Able To Accumulate and Utilize Cholesterol

Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment

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