119
which means the genomic approach is unsuitable for naïve persisters study. One
approach to address these questions is to analyze and compare the global expression
profiles of the persister cells and regular cells. This approach has been applied
exclusively to study E. coli, Acinetobacter baumannii, M. tuberculosis and
Salmonella. The transcriptomic studies have previously mainly relied on microarray
technology. To date, with the development of next generation sequencing technologies, RNA sequencing (RNA-seq) is now the mainstream technology to analyze the
entire transcriptome of persisters.
The first transcriptomic study of persisters was performed by the Lewis group.
They treated the E. coli culture with high concentration of ampicillin, which caused
lysis of regular cells, and collected the surviving persisters by centrifugation
(Fig. 5.2b). The expression profiles of persister cells were determined using a
microarray. Around 300 genes were over expressed in the persister cells, including
toxin-antitoxin (TA) modules and other genes that can block important cellular
functions such as translation and replication (Keren et al. 2004b). By comparing the
expression profiles of ampicillin treatment induced persisters with ofloxacin isolated persisters, 22 common genes were induced, and 139 common genes were
repressed in both groups. The transcriptionally activated genes in both antibiotics
treatment induced persisters encoding ribosomal, stress response and regulatory
proteins, most of the genes repressed are involved in energy metabolism, protein
synthesis and small molecules transportation. Genes commonly changed in different antibiotic isolated persisters might be involved in the conserved persisters formation pathways (Kaldalu et al. 2004). By using RNA-seq, 943 genes were identified
with twofold higher expression in the ceftazidime isolated A. baumannii persister
cells compared with the regular cells (Alkasir et al. 2018). D-cycloserine is an anti
Tuberculosis drug, which can lyse M. tuberculosis effectively. The transcriptome
analysis of D-cycloserine treatment induced M. tuberculosis persisters characterized 282 upregulated genes and 1408 downregulated genes for at least for twofold
(Keren et al. 2011).
Use of bactericidal antibiotics to kill the regular cells within the population represents a very straightforward method to isolate the persister cells. However, the
presence of antibiotics might induce the change of genes unrelated to persisters
formation. Moreover, the global transcriptomic approaches provide an average
value for gene expression over the survived population, which cannot provide information on pre-existing persisters. One approach to isolate pre-existing persisters is
the combination of fluorescent activated cell sorting (FACS) with fluorescent based
persister reporters. Using this approach, pre-existing persisters from the E. coli
planktonic cultures were isolated for the transcriptomic analysis (Henry and
Brynildsen 2016; Jain et al. 2016; Matsumoto et al. 2018; Shah et al. 2006). The
analysis of gene expression profile by this approach has the potential to enlarge our
understanding of the mechanisms involved in persister formation.
Recently, clustered regularly interspaced short palindromic repeats (CRISPR)
approaches have also been applied to study the bacterial persisters (Leung et al.
2015). Other prospect regarding to further address the transcriptional and post transcriptional regulation includes using CRISPRi (CRISPR interference) (Lee et al.
5 Molecular and Systems Biology Approaches for Analyzing Drug-Tolerant Bacterial…
which means the genomic approach is unsuitable for naïve persisters study. One
approach to address these questions is to analyze and compare the global expression
profiles of the persister cells and regular cells. This approach has been applied
exclusively to study E. coli, Acinetobacter baumannii, M. tuberculosis and
Salmonella. The transcriptomic studies have previously mainly relied on microarray
technology. To date, with the development of next generation sequencing technologies, RNA sequencing (RNA-seq) is now the mainstream technology to analyze the
entire transcriptome of persisters.
The first transcriptomic study of persisters was performed by the Lewis group.
They treated the E. coli culture with high concentration of ampicillin, which caused
lysis of regular cells, and collected the surviving persisters by centrifugation
(Fig. 5.2b). The expression profiles of persister cells were determined using a
microarray. Around 300 genes were over expressed in the persister cells, including
toxin-antitoxin (TA) modules and other genes that can block important cellular
functions such as translation and replication (Keren et al. 2004b). By comparing the
expression profiles of ampicillin treatment induced persisters with ofloxacin isolated persisters, 22 common genes were induced, and 139 common genes were
repressed in both groups. The transcriptionally activated genes in both antibiotics
treatment induced persisters encoding ribosomal, stress response and regulatory
proteins, most of the genes repressed are involved in energy metabolism, protein
synthesis and small molecules transportation. Genes commonly changed in different antibiotic isolated persisters might be involved in the conserved persisters formation pathways (Kaldalu et al. 2004). By using RNA-seq, 943 genes were identified
with twofold higher expression in the ceftazidime isolated A. baumannii persister
cells compared with the regular cells (Alkasir et al. 2018). D-cycloserine is an anti
Tuberculosis drug, which can lyse M. tuberculosis effectively. The transcriptome
analysis of D-cycloserine treatment induced M. tuberculosis persisters characterized 282 upregulated genes and 1408 downregulated genes for at least for twofold
(Keren et al. 2011).
Use of bactericidal antibiotics to kill the regular cells within the population represents a very straightforward method to isolate the persister cells. However, the
presence of antibiotics might induce the change of genes unrelated to persisters
formation. Moreover, the global transcriptomic approaches provide an average
value for gene expression over the survived population, which cannot provide information on pre-existing persisters. One approach to isolate pre-existing persisters is
the combination of fluorescent activated cell sorting (FACS) with fluorescent based
persister reporters. Using this approach, pre-existing persisters from the E. coli
planktonic cultures were isolated for the transcriptomic analysis (Henry and
Brynildsen 2016; Jain et al. 2016; Matsumoto et al. 2018; Shah et al. 2006). The
analysis of gene expression profile by this approach has the potential to enlarge our
understanding of the mechanisms involved in persister formation.
Recently, clustered regularly interspaced short palindromic repeats (CRISPR)
approaches have also been applied to study the bacterial persisters (Leung et al.
2015). Other prospect regarding to further address the transcriptional and post transcriptional regulation includes using CRISPRi (CRISPR interference) (Lee et al.
5 Molecular and Systems Biology Approaches for Analyzing Drug-Tolerant Bacterial…