Abstract
Hydrogen peroxide (H2O2) is one of a variety of reactive oxygen species (ROS) produced by aerobic organisms. Host production of toxic H2O2 in response to pathogen infection is an important classical innate defense mechanism against invading microbes. Understanding the mechanisms by which pathogens, in response to oxidative stress, mediate defense against toxic ROS, can reveal anti-microbial targets and shed light on pathogenic mechanisms. In this study, we provide evidence that a Mycobacterium smegmatis hemerythrin-like protein MSMEG_2415, designated MsmHr, is a H2O2-modulated repressor of the SigF-mediated response to H2O2. Circular dichroism and spectrophotometric analysis of MsmHr revealed properties characteristic of a typical hemerythrin-like protein. An msmHr knockout strain of M. smegmatis mc2155 (ΔmsmHr) was more resistant to H2O2 than its parental strain, and overexpression of MsmHr increased mycobacterial susceptibility to H2O2. Mutagenesis studies revealed that the hemerythrin domain of MsmHr is required for the regulation of the H2O2 response observed in the overexpression study. We show that MsmHr inhibits the expression of SigF (MSMEG_1804), an alternative sigma factor that plays an important role in bacterial oxidative stress responses, including those elicited by H2O2, thus providing a mechanistic link between ΔmsmHr and its enhanced resistance to H2O2. Together, these results strongly suggest that MsmHr is involved in the response of mycobacteria to H2O2 by negatively regulating a sigma factor, a function not previously described for hemerythrins.
Highlights
Hydrogen peroxide (H2O2) is a universal oxidative stress molecule produced by aerobic organisms from all three domains of life (Imlay, 2008)
We show that MsmHr inhibits the expression of SigF (MSMEG_1804), an alternative sigma factor that plays an important role in bacterial oxidative stress responses, including those elicited by H2O2, providing a mechanistic link between msmHr and its enhanced resistance to H2O2
Using a genetic and biochemical approach, we show that (i) MsmHr displays circular dichroism (CD) and UVvis spectrophotometric features typical of a hemerythrin-like protein; (ii) relative to wild-type bacilli, the msmHr knockout strain is more resistant to H2O2 and mc2155 overexpressing MsmHr exhibits enhanced H2O2 susceptibility; (iii) the H2O2 response is dependent on the hemerythrin domain; (iv) MsmHr represses sigF transcription through the promoter Prbsw, and participates in regulating the SigF-mediated H2O2 response
Summary
Hydrogen peroxide (H2O2) is a universal oxidative stress molecule produced by aerobic organisms from all three domains of life (Imlay, 2008). The production of H2O2 by the host in response to pathogen infection is an important innate defense mechanism (Fang, 2004) This ROS can be damaging via direct toxic effects or disruption of redox balance, the latter being critical for metabolic homeostasis and survival. The tubercle bacillus, expresses DosS and DosT (Kumar et al, 2007), two sensor histidine kinases whose heme iron plays a critical role in their response to the levels of O2, nitric oxide (NO) and carbon monoxide (CO). Interaction with these various gasses activates the kinase activity of DosS and DosT, relaying the signals to the response regulator DosR. The DosS-DosT/WhiB3 paradigm underscores the importance of iron-containing proteins in the response of M. tuberculosis to redox signals, including those imposed by reactive oxygen intermediates
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