The H2O2 inherently released by the mycobacterial minor subpopulation enhances the survival of the major kin subpopulation against rifampicin

Rashmi Ravindran Nair; Deepti Sharan; Vijay Srinivasan; Nagaraja Mukkayyan; Kishor Jakkala; Parthasarathi Ajitkumar

doi:10.1016/j.crmicr.2022.100148

The H₂O₂ inherently released by the mycobacterial minor subpopulation enhances the survival of the major kin subpopulation against rifampicin

Curr Res Microb Sci. 2022 Jun 18:3:100148. doi: 10.1016/j.crmicr.2022.100148. eCollection 2022.

Authors

Rashmi Ravindran Nair^{1

2}, Deepti Sharan^{1

3}, Vijay Srinivasan^{1

4}, Nagaraja Mukkayyan^{1

5}, Kishor Jakkala^{1

6}, Parthasarathi Ajitkumar¹

Affiliations

¹ Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, Karnataka, India.
² Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
³ Department of Microbiology, University of Chicago, Chicago, IL 60637, USA.
⁴ School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK.
⁵ Department of Microbial Pathogenesis, University of Maryland, Baltimore 21201, Maryland, USA.
⁶ Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.

Abstract

Exposure to antibiotics most often generates oxidative stress in bacteria. Oxidative stress survival mechanisms would facilitate the evolution of antibiotic resistance. As part of an effort to understand oxidative stress survival mechanisms in mycobacteria, here we show that the minor subpopulation (SCs; short-sized cells constituting 10% of the population) of Mycobacterium smegmatis significantly increased the survival of its major kin subpopulation (NCs; normal/long-sized cells constituting 90% of the population) in the mid-log-phase (MLP) cultures against the oxidative stress induced by rifampicin and exogenously added H₂O₂ (positive control). We had earlier shown that the SCs in the MLP cultures inherently and naturally release significantly high levels of H₂O₂ into the medium. Addition of the SCs' culture supernatant, unlike the supernatant of the dimethylthiourea (H₂O₂ scavenger) exposed SCs, enhanced the survival of NCs. It indicated that NCs' survival required the H₂O₂ present in the SCs' supernatant. This H₂O₂ transcriptionally induced high levels of catalase-peroxidase (KatG) in the NCs. The naturally high KatG levels in the NCs significantly neutralised the endogenous H₂O₂ formed upon exposure to rifampicin or H₂O₂, thereby enhancing the survival of NCs against oxidative stress. The absence of such enhanced survival in the furA-katG and katG knockout (KO) mutants of NCs in the presence of wild-type SCs, confirmed the requirement of the H₂O₂ present in the SCs' supernatant and NCs' KatG for enhanced oxidative stress survival. The presence of SCs:NCs at 1:9 in the pulmonary tuberculosis patients' sputum alludes to the clinical significance of the finding.

Keywords: AES,, allelic exchange substrates; Antibiotic stress; Bacterial subpopulations; CFU,, colony forming unit; DMTU,, dimethylthiourea; GFP,, green fluorescence protein; H2O2; HRP,, horse radish peroxidase; Isoniazid; LB,, Luria-Bertani; MLP,, Mid-log-phase; Mycobacteria; NCF,, NCs-enriched fraction; NCs,, normal/long-sized cells; NLP,, Natural-like Proportion; Oxidative stress; PCR,, polymerase chain reaction; ROS,, reactive oxygen species; Rifampicin; SCF,, SCs-enriched fraction; SCs,, short-sized cells; Survival.