The response of mycobacterium tuberculosis to reactive oxygen and nitrogen species

Abstract

The bacteriostatic and bactericidal effects and the transcriptional response of Mycobacterium tuberculosis to representative oxidative and nitrosative stresses were investigated by growth and survival studies and whole genome expression analysis. The M. tuberculosis reaction to a range of hydrogen peroxide (H(2)O(2)) concentrations fell into three distinct categories: (1) low level exposure resulted in induction of a few highly sensitive H(2)O(2)-responsive genes, (2) intermediate exposure resulted in massive transcriptional changes without an effect on growth or survival, and (3) high exposure resulted in a muted transcriptional response and eventual death. M. tuberculosis appears highly resistant to DNA damage-dependent, mode-one killing caused by low millimolar levels of H(2)O(2) and only succumbs to overwhelming levels of oxidative stress observed in mode-two killing. Nitric oxide (NO) exposure initiated much the same transcriptional response as H(2)O(2). However, unlike H(2)O(2) exposure, NO exposure induced dormancy-related genes and caused dose-dependent bacteriostatic activity without killing. Included in the large shared response to H(2)O(2) and NO was the induction of genes encoding iron-sulfur cluster repair functions including iron acquisition. Stress regulons controlled by IdeR, Sigma H, Sigma E, and FurA comprised a large portion of the response to both stresses. Expression of several oxidative stress defense genes was constitutive, or increased moderately from an already elevated constitutive level, suggesting that bacilli are continually primed for oxidative stress defense.

Keywords: Mycobacterium tuberculosis; hydrogen peroxide; microarray; nitric oxide; reactive nitrogen species; reactive oxygen species.

Figure 1

The effect of H₂O₂ stress on growth and survival. (A) Growth of Mtb for 72 h post exposure to H₂O₂ Filled circles, H₂O control, filled squares, 0.05; filled triangles 0.5, filled downward triangles, 5.0: filled diamonds, 10.0; open circles 50; and open squares 200 mM H₂O₂ (B) Survival of Mtb monitored by colony forming units 4 h, white bars, and 24 h, gray bars, hours post exposure to a range of H₂O₂ doses between 0.05 and 200 mM.

Figure 2

The effect of NO stress on growth and survival. (A) Growth of Mtb for 96 h post exposure to slow release NO donor DETA/NO. Filled circles, 0.01 N NaOH control; filled squares, 0.005: filled triangles 0.05; filled downward triangles, 0.5; filled diamonds, 1.0; and open circles 5 mM DETA/NO (B) Survival of Mtb over a range of low starting cell densities (0.01, 0.001, and 0.0001 OD) monitored by colony forming units. White bars indicate 0.01 N NaOH controls and gray bars indicate exposure to 5 mM of the rapid release NO donor spermidine-NONOate Time points were taken before addition of NO donor (0) and after 4 h of exposure to NO donor (4).

Figure 3

Distribution of genes regulated in response to NO and H₂O₂. (A) 174 were induced at least 2-fold after 40 min exposure to 0.005, 0.05, 0.5, 1.0, or 5.0 mM DETA/NO and fulfilled the criteria for inclusion in Table A1. Forty-one of the 174 genes are members of the previously published DosR regulon (Voskuil et al., 2003) and are therefore not included in Table A1. A majority of genes were regulated by both NO and H₂O₂ and 53 genes were regulated solely by NO including the 41 the DosR regulon genes (B) 163 genes from Table A1 were induced by H₂O₂, of these 42 were not induced by NO. Four transcriptional regulators: SigH (Imlay, 2003), IdeR (Rodriguez et al., 2002), SigH (Park and Imlay, 2003), and FurA (this study and Pym et al., ; Zahrt et al., 2001) control genes that encompass 33% of the H₂O₂ response.