The rifampicin-inactivating mono-ADP-ribosyl transferase ofMycobacterium smegmatissignificantly influences reactive oxygen species levels in the actively growing cells

Abstract

A classic example of antibiotic inactivating function in bacteria is the Mycobacterium smegmatis (Msm) encoded rifampicin-inactivating mono-ADP-ribosyl transferase (arr). Since its probable biological role has been proposed to be in DNA damage response, which is inflicted by reactive oxygen species (ROS), in the present study, we examined whether Msm Arr influences ROS levels. For this purpose, the levels of the ROS, hydroxyl radical and superoxide, were determined in the mid-log phase (MLP) cells of Msm arr knockout (arr-KO) strain, in comparison to those in the equivalently grown Msm arr+ wild-type (WT) strain. The MLP arr-KO cells generated significantly elevated levels of superoxide and hydroxyl radical, unlike the equivalently grown WT MLP cells. Complementation of arr-KO with arr, but not with empty vector, restored the ROS levels comparable to those in the WT strain. Elevated ROS levels in the arr-KO strain enabled selection of rifampicin-resistant mutants at 10-7 cfu/ml from the rifampicin-unexposed MLP cells of arr-KO, which is one-log10 higher than that for WT cells (10-8). Upon prolonged exposure to rifampicin, the susceptibility, persister formation, generation of elevated levels of hydroxyl radical by the persisters, rifampicin-resister generation frequency of the persisters and regrowth of the rifampicin-resistant mutants from the respective persisters were all comparable between the arr-KO and WT strains. These observations revealed that Arr influences ROS levels in the actively growing M. smegmatis cells but not in the rifampicin-exposed cells. We proposed the probable pathway through which Arr might be influencing ROS levels in the actively growing M. smegmatis cells.IMPORTANCEDiverse genera of bacteria consisting of pathogens, opportunistic pathogens and non-pathogens, possess Arr-type activities that confer equally efficient rifampicin resistance, thereby posing serious health hazard. Acquisition of this function by other bacteria through horizontal gene transfer enhances the hazard posed by the bacteria possessing it. M. smegmatis is an opportunistic human pathogen that causes infections of skin and soft tissues. Moreover, M. smegmatis is a genetically tractable model organism for M. tuberculosis with the potential to function even as tuberculosis vaccine. In view of these significant aspects of Arr and M. smegmatis, the study to find out the natural physiological role of Arr in M. smegmatis, gains importance for designing strategies to prevent antibiotic inactivation and to target the cellular function to contain the bacterium. Above all, the three-dimensional structure of M. smegmatis Arr reveals significant structural homology with eukaryotic ADP-ribosyltransferases and bacterial toxins, thereby giving the study broad significance.

Unique Mode of Cell Division by the Mycobacterial Genetic Resister Clones Emerging De Novo from the Antibiotic-Surviving Population

“…Precursors lethal concentrations of rifampicin/moxifloxacin, as demonstrated by us in the present study and earlier (9)(10)(11). It implied that these subpopulations contained rifampicintolerant cells, as defined (16), classical persisters, as termed (4,5), and several other phenotypic subpopulations that might survive using several other mechanisms (2, 7).…”

“…We used 10ϫ MBC rifampicin, in addition to 1ϫ MBC rifampicin, for exposure because M. smegmatis, unlike M. tuberculosis, has ADP-ribosyl transferase (Arr) that inactivates rifampicin (17). We have confirmed that 10ϫ MBC rifampicin inflicts lethality on M. smegmatis (10 8 cells/ml) despite having Arr (11). Further, 10 6 cells/ml were used for exposure to rifampicin to preempt the emergence of natural rifampicin resisters at ϳ10 Ϫ8 frequency (11,18).…”

“…The third replicate showed resister emergence at 42 h with a resister generation frequency of 3.5 ϫ 10 Ϫ3 . The rifampicin resister generation frequency of ϳ10 Ϫ3 was ϳ5-log 10 -fold higher than the natural resister generation frequency of 10 Ϫ8 of M. smegmatis against rifampicin (11,18). The high resister generation frequency alluded to elevated levels of the DNA-nonspecific mutagen, hydroxyl radical (20)(21)(22), as found by us in the rifampicin/moxifloxacin-exposed M. tuberculosis and M. smegmatis antibioticsurviving cells (9)(10)(11) and in the rifampin exposed M. tuberculosis cells (23) and by others in other bacterial systems exposed to antibiotics (12)(13)(14).…”

“…6A, red line). These resister generation frequencies were ϳ2 to 3 log 10 -fold higher than the natural resister generation frequency of 10 Ϫ8 of M. smegmatis against rifampicin (11,18) and about ϳ2 to 3 log 10 -fold lower than that of the 10ϫ MBC rifampicin cultures (compare Fig. S4F with Fig.…”

A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

“…Hydroxyl radical being a mutagen without nucleotide sequence specificity (Imlay et al, 1988; Sakai et al, 2006), we examined the benefits of its differential levels between the SCF and the NCF cells in terms of resister generation frequency against two anti-tuberculosis antibiotics, rifampicin and moxifloxacin, which have completely different targets of action. For this purpose, the freshly fractionated Msm SCF and NCF cells were entirely plated on Middlebrook 7H10 agar containing rifampicin (125 μg/ml; 3× MBC; Swaminath, 2017) or moxifloxacin (0.5 μg/ml; 5× MBC; Swaminath, 2017). The colonies from the rifampicin and moxifloxacin plates showed mutations in the rifampicin resistance determining region (RRDR) and quinolone resistance determining region (QRDR) for rifampicin and moxifloxacin resistance, respectively (Supplementary Figures S3, S4, respectively; Takiff et al, 1994; Yue et al, 2003; Chien et al, 2016).…”