Identification of a Chemical That Inhibits the Mycobacterial UvrABC Complex in Nucleotide Excision Repair

Abstract

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen’s ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb’s Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

“…10-fold lower survival than wild-type after a 40 mJ/cm 2 dose (Figure 7A). The Δ rnhB stationary phase UV phenotype was much milder than that of a stationary phase Δ uvrB strain that lacks the UV damage recognition protein UvrB (23) or a stationary phase Δ uvrD1 strain that lacks the UvrD1 helicase component of the mycobacterial nucleotide excision repair pathway (Figure 7A) (24). The Δ rnhA Δ rnhB double mutant was more sensitive than Δ rnhB to killing by UV in stationary phase (Figure 7B).…”

A Comparative Study of the Bactericidal Activity and Daily Disinfection Housekeeping Surfaces by a New Portable Pulsed UV Radiation Device

“…B. subtilis spores) [22], (ii) high catalase activity (Acinetobacter sp.) [9], (iii) sodium chloride concentrations in biofilm (Listeria monocytogenes) [3], (iv) biofilm formation (P. aeruginosa) [10], and (v) nucleotide excision repair by the uvrABC complex (Mycobacterium smegmatis) [19]. Considering the possibility that UV-tolerant bacteria could be present in the environment, such as P. aeruginosa, A. baumannii and B. cereus, as previously described, the pulsed UV radiation is a sufficient UV irradiation dose for disinfection of the environment.…”

Stressed Mycobacteria Use the Chaperone ClpB to Sequester Irreversibly Oxidized Proteins Asymmetrically Within and Between Cells

“…Mtb’s defenses against host-imposed stresses include catabolizing ROS and RNS (Bryk et al, 2002), repairing their damage (Mazloum et al, 2011), and degrading damaged macromolecules that cannot be repaired (Lin et al, 2009). …”