Synthesis and biological activity of some derivatives of rifamycin P

Cavalleri, B.; Turconi, Marco; Tamborini, G.; Occelli, E; Cietto, G.; Pallanza, R; Scotti, Roberto; Berti, Marisa; Romanò, Gabriella; Parenti, Francesco

doi:10.1021/jm00167a029

Cited by 22 publications

(13 citation statements)

References 8 publications

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“…Small amounts of rifampin, a semisynthetic antibiotic of the rifamycin group (6), were shown to induce F of this pathogen (15). Rifampin blocks the ␤ subunit of prokaryotic DNA-dependent RNA polymerase (5,11) and by this mechanism inhibits RNA synthesis at an early stage of elongation.…”

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Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ ^B -Dependent General and Multiple Stress Response

2002

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Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of Bacillus subtilis led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment. L-[35 S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The B -dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the ␤ subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the D regulon were also induced. The rifampin-triggered growth arrest was extended in a sigB mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the B signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The sigB mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the sigB mutant by a factor of 100. We conclude that the general stress response of B. subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.The natural habitat of the gram-positive bacterium Bacillus subtilis is the soil-an environment in which stress and starvation are the rule. Not surprisingly B. subtilis has evolved a range of strategies to survive under these hostile conditions. The formation of stress-resistant spores, for example, guarantees long-term survival, while uptake of external DNA in the process of genetic competence may contribute to adaptation by recombination (12). Besides these time-consuming processes B. subtilis is able to quickly develop short-term protection of nongrowing cells against multiple stress conditions, including acidic, alkaline, osmotic, or oxidative stress, heat, or ethanol. A global regulator, the alternative sigma factor B , controls this general stress response. The B regulon contains about 150 genes, some of which have been shown to actively protect or repair macromolecules (see references 12 and 18 for reviews). Under laboratory conditions B is activated by two different groups of stimuli: physical stresses like heat, acid, or ethanol act via the RsbU-dependent pathway, while B activation after glucose or phosphate starvation is RsbP dependent (20,21,24).An alternative sigma factor homologous to B of B. subtilis also exists in Mycobacterium tuberculosi...

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Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ ^B -Dependent General and Multiple Stress Response

2002

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“…Rifampin is an important chemotherapeutic agent for use against tuberculosis, leprosy, and infections by organisms related to those causing these diseases (3,7). The antimicrobial activity of rifampin is due to its inhibition of DNA-dependent RNA polymerase, and most rifampin-resistant bacteria have been reported to have an alteration in the ␤-subunit of this enzyme (2).…”

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Ribosylation by mycobacterial strains as a new mechanism of rifampin inactivation

Dabbs

Yazawa

Mikami

et al. 1995

Antimicrob Agents Chemother

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Rifampin is an important chemotherapeutic agent for use against tuberculosis, leprosy, and infections by organisms related to those causing these diseases (3, 7). The antimicrobial activity of rifampin is due to its inhibition of DNA-dependent RNA polymerase, and most rifampin-resistant bacteria have been reported to have an alteration in the ␤-subunit of this enzyme (2). Such a resistance mechanism has been reported for Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium africanum, and Mycobacterium leprae (14, 16). During our studies of fast-growing mycobacterial strains, we found that several strains inactivate rifampin (1, 5). We analyzed the inactivated antibiotics and found them to differ in mass spectrum and chromatographic mobility from those previously reported, i.e., the glycosylated (glucosylated) or phosphorylated compounds produced by pathogenic Nocardia spp. (11,17). These results prompted us to determine the detailed structures of the inactivated compounds. In this paper the isolation, structures, and antimicrobial activities of the inactivated compounds are reported.Rifampin was generously provided by CIBA-GEIGY Pharmaceuticals, Basel, Switzerland. MICs were determined by an agar dilution method with brain heart infusion agar (Difco Laboratories, Detroit, Mich.) medium. The inoculum size of each test organism was adjusted to 10 6 CFU/ml. The plates were spotted with a multipoint inoculator (A 400; Denly Instruments, Ltd., Sussex, England) that delivered 0.005 ml of inoculum, resulting in a spot inoculum of approximately 5 ϫ 10 4 CFU. Inactivation of rifampin was monitored by a bioassay method with Bacillus subtilis PCI 219 as a test organism (17). Inactivated compounds were monitored with a thin-layer chromatography scanner (CS-910; Shimadzu Seisakusho, Kyoto, Japan) (17, 18) at 238 nm.One loopful of the culture from slant cultures of Mycobacterium smegmatis DSM 43756 was inoculated into a 100-ml Erlenmeyer shake flask containing 20 ml of a seed culture medium (2% glycerol-enriched brain heart infusion medium; Difco Laboratories) with 4-mm-diameter glass beads to reduce aggregation of the mycobacterial cells (18). The inoculated flasks were shaken at 250 rpm (5.8-cm stroke) for 4 days at 33ЊC. For large-scale preparation of the inactivation products, the seed culture was used as an inoculum. Ten milliliters of seed culture was inoculated into 500-ml shake flasks containing 100 ml of the same medium. After 24 h of incubation at 33ЊC, rifampin (stock solution in methanol at 100 mg/ml) was added to a final concentration of 50 g/ml, and the mixture was incubated for 3 days. Following separation from mycelia by centrifugation at 6,000 rpm (5,800 ϫ g), the supernatant was adjusted to pH 2.0 with 1 N HCl and extracted three times with an equal volume of ethylacetate. The extracts were combined, dehydrated with Na 2 SO 4 , and concentrated in vacuo to dryness. The dried material was purified by silica gel column chromatography (column size, 3.5 by 15 cm) by using 75 g of Wakogel C-200 (Wako Pu...

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“…Consequently, rifampin ( Fig. 1), which is now used as a valuable chemotherapeutic agent, was introduced (2,3,6). Rifampin has activity against a great variety of organisms, such as bacteria, viruses, and eukaryotes.…”

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Inactivation of rifampin by Nocardia brasiliensis

Yazawa

Mikami

Maeda

et al. 1993

Antimicrob Agents Chemother

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Rifampin was glycosylated by a pathogenic species of Nocardia, i.e., Nocardia brasiliensis. The structures of two glycosylated compounds (RIP-1 and RIP-2) isolated from the culture broth of the bacterium were Rifamycins are members of the naphthalenic ansamycin group of antibiotics characterized by a 17-membered ansa chain (1,5,11,17 (Becton Dickinson, Cockeysville, Md.). One loopful of spores or mycelial fragments from the slant cultures of Nocardia spp. was inoculated into a 10-ml Erlenmeyer shake flask containing 5 ml of a seed medium (2% glucose-enriched brain heart infusion medium; Difco Laboratories, Detroit, Mich.) with 4-mm glass beads intended to reduce aggregation of the nocardial cells (21). The inoculated flasks were shaken at 250 rpm (5.8-cm stroke) for 4 days at 27°C. The mature seed cultures were used as inocula for the inactivation or MIC experiments. Other bacterial inocula were prepared by the method previously described (21).MIC determination. The MIC, defined as the lowest drug concentration causing complete inhibition of visible growth, was determined by an agar dilution method using MuellerHinton II agar medium to give final concentrations from 0.39 to 400 ,ug/ml. Drug-free plates were included as a bacterial growth control. The inoculum size of a test organism was adjusted to 107 CFU/ml for Nocardia spp. and 106 CFU/ml for other bacteria. The plates were spotted with a multipoint inoculator (A 400; Denly Instruments, Ltd., Sussex, England) that delivered 0.005 ml, resulting in a spot inoculum of approximately 5 x 104 CFU.Time course of rifampin inactivation. The mature seed cultures were inoculated at a 10% rate into 100-ml Erlenmeyer flasks containing 50 ml of brain heart infusion medium. Methanol-sterilized rifampin was added aseptically at a final concentration of 20 or 200 ,ug/ml. For determination of the rifampin concentration, 2 ml of the flask cultures was harvested at various time intervals and filtered. The pH of the filtrate was adjusted to 2.0 with 2 N HCl, and the filtrate was then extracted with 2 ml of ethyl acetate. After concentration of the extract in vacuo to dryness, 100 ,ul of methanol was added and 50 ,ul of the mixture was tested for antibacterial activity. The rifampin concentration was measured by the size of the inhibition zone on a lawn of Bacillus subtilis PCI 219 grown on nutrient agar (Difco) (15). Inactivation products were monitored by thin-layer chromatography (TLC) using CHCl3-MeOH (4:1) as a developing solvent. Although rifampin or its inactivation products were easily detected by their characteristic reddish brown spots on untreated TLC plates, the inactivation products were usually monitored additionally by a dual-wavelength TLC scanner (CS-91OM; Shimadzu Seisakujyo, Tokyo, Japan) at 238 nm. Growth was determined by measuring the dry weight of mycelia: specifically, 5 ml of the culture was harvested at 1313

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Synthesis and biological activity of some derivatives of rifamycin P

Cited by 22 publications

References 8 publications

Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ ^B -Dependent General and Multiple Stress Response

Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ ^B -Dependent General and Multiple Stress Response

Ribosylation by mycobacterial strains as a new mechanism of rifampin inactivation

Inactivation of rifampin by Nocardia brasiliensis

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Synthesis and biological activity of some derivatives of rifamycin P

Cited by 22 publications

References 8 publications

Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ B -Dependent General and Multiple Stress Response

Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ B -Dependent General and Multiple Stress Response

Ribosylation by mycobacterial strains as a new mechanism of rifampin inactivation

Inactivation of rifampin by Nocardia brasiliensis

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Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ ^B -Dependent General and Multiple Stress Response

Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σ ^B -Dependent General and Multiple Stress Response