Abstract
Reactive nitrogen species (RNS) are secreted by human cells in response to infection by Mycobacterium tuberculosis (Mtb). Although RNS can kill Mtb under some circumstances, Mtb can adapt and survive in the presence of RNS by a process that involves modulation of gene expression. Previous studies focused primarily on stress-related changes in the Mtb transcriptome. This study unveils changes in the Mtb proteome in response to a sub-lethal dose of nitric oxide (NO) over several hours of exposure. Proteins were identified using liquid chromatography coupled with electrospray ionization mass spectrometry (LC–MS/MS). A total of 2911 Mtb proteins were identified, of which 581 were differentially abundant (DA) after exposure to NO in at least one of the four time points (30 min, 2 h, 6 h, and 20 h). The proteomic response to NO was marked by two phases, with few DA proteins in the early phase and a multitude of DA proteins in the later phase. The efflux pump Rv1687 stood out as being the only protein more abundant at all the time points and might play a role in the early protection of Mtb against nitrosative stress. These changes appeared to be compensatory in nature, contributing to iron homeostasis, energy metabolism, and other stress responses. This study thereby provides new insights into the response of Mtb to NO at the level of proteomics.
Highlights
Mycobacterium tuberculosis (Mtb) is a successful human pathogen, with the ability to survive the effects of host− defense mechanisms, including intracellular and secreted antibacterial reactive nitrogen and oxygen species (RNS/ ROS) produced by macrophages
Abundant Proteins in Mtb Cells Exposed to Nitrosative Stress
We identified 33,509 peptides assigned to 2,911 proteins, which accounts for 72.9% of the predicted Mtb proteome (Table S1)
Summary
Mycobacterium tuberculosis (Mtb) is a successful human pathogen, with the ability to survive the effects of host− defense mechanisms, including intracellular and secreted antibacterial reactive nitrogen and oxygen species (RNS/ ROS) produced by macrophages. Mtb-infected macrophages generate nitric oxide (NO) and other RNS via the inducible nitric oxide synthase (iNOS).[1] RNS can be lethal, Mtb is able to protect itself against the devastating effect of RNS by direct scavenging, iron sequestration, suppression of RNS production, catalytic detoxification, and other stress responses, including NO-inducible DosR-dependent repair of RNS-mediated cellular damage.[2]. In response to host-generated RNS/ROS, Mtb upregulates expression of redox-scavenging enzymes, including catalase (KatG), superoxide dismutases (SodA and SodC), and the peroxynitrite reductase and peroxidase complex (PNR-P) encoded by ahpC, ahpD, lpd, and sucB (dlaT).2a,3 In analogy, mice deficient in dlaT expression are hyper-susceptible to RNS.[3] Another defense against RNS involves oxygen-dependent catalytic detoxification of NO, which is mediated by truncated hemoglobin (trHbN) and prevents inhibition of aerobic respiration by NO. Exposure to NO-induced stress upregulates expression of alpha crystalline (Acr), bacterioferritin (BfrB), and the DosR regulon in Mtb; little is known about the role of these Mtb genes/proteins in the response to nitrosative stress.2a
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