Dataset Information

Associating H2O2 and NO related changes in the proteome of Mycobacterium smegmatis with enhanced survival in macrophages

ABSTRACT: In this study, we used mass spectrometry-based discovery proteomic analysis to examine the cell-wide changes of Mycobacterium smegmatis, in response to sub-lethal concentrations of H2O2 and nitric oxide (NO) over time. Preliminary results have shown that sub-lethal concentrations of reactive oxygen and nitrogen species may act as signalling molecules by playing a role in the overall protein abundance that may be conducive to survival in vivo. The presented study showed that both treatments induced: the DosR regulon, alterations in protein expression and lipid metabolism. In addition to the proteomic changes observed, we have also observed increased survival after macrophage infection of Mycobacterium smegmatis pre-exposed to sub-lethal doses of H2O2 or DETA-NO. ¬ In total, 27 samples yielded approximately 3300 M. smegmatis protein group identifications. More in-depth analysis of this new dataset will provide a better understanding as to the inner workings of mycobacteria under oxidative and nitrosative stress and potential mechanisms by which exposure to sub-lethal concentrations of stressors results in an increased resistance to stress.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mycobacterium Smegmatis

TISSUE(S): Cell Culture

SUBMITTER: Bridget Calder

LAB HEAD: Jonathan Blackburn

PROVIDER: PXD010020 | Pride | 2019-11-12

REPOSITORIES: Pride

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			Action	DRS
	150113_Jess_DETA_T1B1_200ng_ul.raw	Raw
	150113_Jess_DETA_T1B2_200ng_ul.raw	Raw
	150113_Jess_DETA_T1B3_200ng_ul_150119035315.raw	Raw
	150113_Jess_DETA_T2B1_200ng_ul.raw	Raw
	150113_Jess_DETA_T2B2_200ng_ul.raw	Raw

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Associating H<sub>2</sub>O<sub>2-</sub>and NO-related changes in the proteome of Mycobacterium smegmatis with enhanced survival in macrophage.

Ganief Naadir N Sjouerman Jessica J Albeldas Claudia C Nakedi Kehilwe C KC Hermann Clemens C Calder Bridget B Blackburn Jonathan M JM Soares Nelson C NC

Emerging microbes & infections 20181213 1

Mycobacterium manages to evade the host cell immune system, partially owing to its ability to survive redox stress after macrophage engulfment. Exposure to redox stress has been linked to later replication, persistence, and latent infection. In this work, mass spectrometry was used to elucidate the cell-wide changes that occur in response to sublethal doses of hydrogen peroxide and nitric oxide over time, with Mycobacterium smegmatis being used as a model organism. A total of 3135 proteins were ...[more]

PMID: 30546046

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Project description:Oxidative stress (OS) results from genetic defects or stressful environmental challenges that cause unchecked production of reactive oxygen species (ROS). OS has been implicated in many diseases due to its wide ranging damage to nucleic acids, proteins and lipids. Using Halobacterium salinarum NRC-1, an extremophile that thrives under conditions of excessive OS, we have constructed a systems model for oxidative stress response (OSR) by integrating transcriptional changes induced by treatments with H2O2 and paraquat, functional associations inferred through comparative genomics, and de novo discovered cis-regulatory motifs. Together with phenotypic analysis of mutant strains this has revealed a multi-tiered OS management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids, and gas vesicles, metal trafficking, and various other aspects of metabolism. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was previously constructed from cellular responses to diverse environmental perturbations –this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of g rays. Mid-log phase cultures of H. salinarum NRC-1 (2 biological replicates) grown in flasks were treated with either sub-lethal concentrations of 25mM H2O2 or 4mM PQ and incubated with shaking for up to 240 min. For each treatment, two time courses were run to determine the transcriptional responses to (1) constant stress and (2) recovery of H. salinarum NRC-1 to H2O2 and PQ. During constant stress, culture aliquots (~4ml) were collected over a time course (-1, 5, 10, 20, 40, 80 and 160 minutes), centrifuged (16000g, 90 seconds) and flash frozen. For recovery, cells were first treated for 2-hours with either 25 mM H2O2 or 4mM PQ, recovered by centrifugation, washed and re-suspended in CM. Cultures were returned to the incubator with shaking and samples were taken at 0, 10, 20, 30, 40, 50, 60, 120, and 240 minutes and processed as described previously. Analysis of temporal transcriptional changes (-1, 0, 5, 10, 20, 40, 80, 160 and 320m) to sub-inhibitory concentrations of PQ (0.25mM) was also characterized. Total RNA was prepared and labeled with Alexa547 and Alexa647 dyes. Intensities were normalized between the Alexa 546 and Alexa 647 channels by scaling all intensities in one channel such that the 75th percentile in each channel was made equal.