Characterization of a Rifampin-Inactivating Glycosyltransferase from a Screen of Environmental Actinomycetes

Abstract

Identifying and understanding the collection of all antibiotic resistance determinants presented in the global microbiota, the antibiotic resistome, provides insight into the evolution of antibiotic resistance and critical information for the development of future antimicrobials. The rifamycins are broad-spectrum antibiotics that target bacterial transcription by inhibition of RNA polymerase. Although mutational alteration of the drug target is the predominant mechanism of resistance to this family of antibiotics in the clinic, a number of diverse inactivation mechanisms have also been reported. In this report, we investigate a subset of environmental rifampin-resistant actinomycete isolates and identify a diverse collection of rifampin inactivation mechanisms. We describe a single isolate, WAC1438, capable of inactivating rifampin by glycosylation. A draft genome sequence of WAC1438 (most closely related to Streptomyces speibonae, according to a 16S rRNA gene comparison) was assembled, and the associated rifampin glycosyltransferase open reading frame, rgt1438, was identified. The role of rgt1438 in rifampin resistance was confirmed by its disruption in the bacterial chromosome, resulting in a loss of antibiotic inactivation and a 4-fold decrease in MIC. Interestingly, examination of the RNA polymerase ␤-subunit sequence of WAC1438 suggests that it harbors a resistant target and thus possesses dual mechanisms of rifamycin resistance. Using an in vitro assay with purified enzyme, Rgt1438 could inactivate a variety of rifamycin antibiotics with comparable steady-state kinetics constants. Our results identify rgt1438 as a rifampin resistance determinant from WAC1438 capable of inactivating an assortment of rifamycins, adding a new element to the rifampin resistome.

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase

“…At least four RIF-deactivating enzymes have been described: ADP-ribosyltransferase (Arr) (6), glycosyltransferase (Rgt) (7,8), phosphotransferase (Rph) (8 -11), and RIF monooxygenase (12, 13). Arr and Rgt act on a critical hydroxyl group (C23) located on the ansa aliphatic chain of RIF, whereas Rph adds a phosphate group to the C21 hydroxyl.…”

“…Covalent modification of RIF hydroxyls with ADP-ribose or phosphate results in high level resistance in Escherichia coli, such as a 64-fold increase in the RIF minimal inhibition concentration (6,10). Modification of RIF by Rgt results in a 4-fold increase of Streptomyces speibonae cultures (7) and markedly reduces the activity against Gram-positive bacteria (8).…”

“…1) is a potent frontline antibiotic against tuberculosis and other mycobacterial infections, but extensive usage of RIF and its derivatives has contributed to bacterial resistance, which neutralizes antibiotic activity (1, 2). In addition to the point mutations in RNA polymerase that are responsible for resistance in mycobacteria (3, 4), some bacterial species, such as soil actinomycetes and parasitic bacteria, employ secondary enzyme-mediated inactivation mechanisms that chemically modify RIF to less active forms or degradation products (5).At least four RIF-deactivating enzymes have been described: ADP-ribosyltransferase (Arr) (6), glycosyltransferase (Rgt) (7,8), phosphotransferase (Rph) (8 -11), and RIF monooxygenase (12, 13). Arr and Rgt act on a critical hydroxyl group (C23) located on the ansa aliphatic chain of RIF, whereas Rph adds a phosphate group to the C21 hydroxyl.…”

Structural basis of rifampin inactivation by rifampin phosphotransferase

“…In M. tuberculosis and other mycobacteria the most common resistance mechanisms are point mutations of the target, the RNAP β-subunit; these mutations significantly decrease the binding of rifamycins and thus neutralize the antibiotic activity (10). Another prevalent resistance strategy adopted by bacteria is modification of the rifamycins, such as ADP ribosylation, glycosylation, and phosphorylation (11)(12)(13). These covalent modifications occur on the critical hydroxyls of the 1-amino, 2-naphthol, 4-sulfonic acid (ansa) chain of rifamycins and thus make rifamycins unable to fit into the binding pocket on RNAP.…”

Arr-cb Is a Rifampin Resistance Determinant Found Active or Cryptic in Clostridiumbolteae Strains

“…In addition, three enzymatic inactivation mechanisms attenuating the affinity of rifampin for the ␤ subunit of RNA polymerase have been reported. Rifampin glycosylation and phosphorylation have been found initially in actinomyces (7)(8)(9), while ADP ribosylation is widely distributed on the basis of the presence of genes encoding predicted Arr enzymes in various pathogenic and nonpathogenic bacteria (10). Three proteins have been shown to be able to catalyze the ADP ribosylation of rifampin.…”