120
2019a) to investigate the mechanisms of the persisters within the life cycle of bacterial cell dynamically.
5.5.3 Proteomics and Metabolomics Approaches
for Persisters Studies
Two dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is a potent tool
to analyse the global protein expression of bacteria. 2D PAGE separates the whole
cell protein extractions into protein spots. The differential protein spots can be cut
from the gel and processed for the liquid chromatography/mass spectrometry analysis (Lilley and Friedman 2004). 2D PAGE has been used in several studies of persisters proteomes. For example, Starck et al. used 2D PAGE to characterize the
non-replicating M. tuberculosis under anaerobic condition (Starck et al. 2004). 2D
PAGE has also been used to characterize the changes in proteome patterns of starvation induced M. tuberculosis persisters over time (Betts et al. 2002). One of the
highly upregulated gene Rv3290c (encoding a lysine-e aminotransferase) has been
demonstrated to be involved in mycobacteria persister formation (Duan et al. 2016;
Lee et al. 2019b). Albrethsen et al. used label free LC-MS/MS (liquid
chromatography- mass spectrometry/mass spectrometry) and two dimensional
DIGE (differential gel electrophoresis) and observed that most of the toxin–antitoxin systems (TAs) were up-regulated in the non-replicating state (Albrethsen et al.
2013). 2D PAGE still has limitations in global protein measurement owing to the
low protein coverage, sensitivity, dynamic range, and precision (Gygi et al. 2000).
For the shotgun proteomics analysis, complex proteins were digested with specific
protease, such as trypsin, the ensuing peptide mixture then analyzed by LC-MS
(Wolters et al. 2001). Label free approaches including isotope coded affinity tag
based and SWATH mass spectrometry based methods have been developed and
applied to non-replicating and reactivated M. tuberculosis samples (Cho et al. 2006;
Gopinath et al. 2015; Schubert et al. 2015). Protein samples from antibiotics isolated E. coli (Sulaiman et al. 2018) and Porphyromonas gingivalis (Li et al. 2018)
persisters have been analyzed by LC-MS which revealed that the membrane integrity and cellular redox state are important for persisters formation.
Proteomics has also been applied to study the antibiotic tolerant bacterial populations in P. aeruginosa biofilms. It has been previously reported that the antibiotic
tolerant bacterial populations in P. aeruginosa biofilms are persister cells (Brooun
et al. 2000; Spoering and Lewis 2001). By means of bio-orthogonal noncanonical
amino acid tagging (BONCAT) proteomic analysis method, Babin et al. illustrated
that flagellar motility and purine synthesis are critical for antibiotic tolerant persisters formation (Babin et al. 2017). In the other study, Chua et al. treated P. aeruginosa biofilms with colistin for 48 h, washed the cells, collected the persister fractions
and investigated their proteomic profile by pulsed stable isotope labelling with
amino acids (pulsed-SILAC) method. P. aeruginosa biofilms were cultivated in
X. Duan et al.
2019a) to investigate the mechanisms of the persisters within the life cycle of bacterial cell dynamically.
5.5.3 Proteomics and Metabolomics Approaches
for Persisters Studies
Two dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is a potent tool
to analyse the global protein expression of bacteria. 2D PAGE separates the whole
cell protein extractions into protein spots. The differential protein spots can be cut
from the gel and processed for the liquid chromatography/mass spectrometry analysis (Lilley and Friedman 2004). 2D PAGE has been used in several studies of persisters proteomes. For example, Starck et al. used 2D PAGE to characterize the
non-replicating M. tuberculosis under anaerobic condition (Starck et al. 2004). 2D
PAGE has also been used to characterize the changes in proteome patterns of starvation induced M. tuberculosis persisters over time (Betts et al. 2002). One of the
highly upregulated gene Rv3290c (encoding a lysine-e aminotransferase) has been
demonstrated to be involved in mycobacteria persister formation (Duan et al. 2016;
Lee et al. 2019b). Albrethsen et al. used label free LC-MS/MS (liquid
chromatography- mass spectrometry/mass spectrometry) and two dimensional
DIGE (differential gel electrophoresis) and observed that most of the toxin–antitoxin systems (TAs) were up-regulated in the non-replicating state (Albrethsen et al.
2013). 2D PAGE still has limitations in global protein measurement owing to the
low protein coverage, sensitivity, dynamic range, and precision (Gygi et al. 2000).
For the shotgun proteomics analysis, complex proteins were digested with specific
protease, such as trypsin, the ensuing peptide mixture then analyzed by LC-MS
(Wolters et al. 2001). Label free approaches including isotope coded affinity tag
based and SWATH mass spectrometry based methods have been developed and
applied to non-replicating and reactivated M. tuberculosis samples (Cho et al. 2006;
Gopinath et al. 2015; Schubert et al. 2015). Protein samples from antibiotics isolated E. coli (Sulaiman et al. 2018) and Porphyromonas gingivalis (Li et al. 2018)
persisters have been analyzed by LC-MS which revealed that the membrane integrity and cellular redox state are important for persisters formation.
Proteomics has also been applied to study the antibiotic tolerant bacterial populations in P. aeruginosa biofilms. It has been previously reported that the antibiotic
tolerant bacterial populations in P. aeruginosa biofilms are persister cells (Brooun
et al. 2000; Spoering and Lewis 2001). By means of bio-orthogonal noncanonical
amino acid tagging (BONCAT) proteomic analysis method, Babin et al. illustrated
that flagellar motility and purine synthesis are critical for antibiotic tolerant persisters formation (Babin et al. 2017). In the other study, Chua et al. treated P. aeruginosa biofilms with colistin for 48 h, washed the cells, collected the persister fractions
and investigated their proteomic profile by pulsed stable isotope labelling with
amino acids (pulsed-SILAC) method. P. aeruginosa biofilms were cultivated in
X. Duan et al.