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Transcriptional Profiling of Mycobacterium tuberculosis Exposed to In

Vitro Lysosomal Stress
Wenwei Lin,a,b,c* Paola Florez de Sessions,d Garrett Hor Keong Teoh,d Ahmad Naim Nazri Mohamed,d Yuan O. Zhu,d
Vanessa Hui Qi Koh,a,b Michelle Lay Teng Ang,a,b* Peter C. Dedon,c Martin Lloyd Hibberd,d,e Sylvie Alonsoa,b,c
Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singaporea; Immunology Programme, Life Sciences
Institute, National University of Singapore, Singaporeb; Infectious Disease Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology,
Singaporec; Genome Institute of Singapore, Agency for Science, Technology and Research, Singapored; Department of Pathogen Molecular Biology, London School of
Hygiene & Tropical Medicine, London, United Kingdome

Increasing experimental evidence supports the idea that Mycobacterium tuberculosis has evolved strategies to survive within
lysosomes of activated macrophages. To further our knowledge of M. tuberculosis response to the hostile lysosomal environ-
ment, we profiled the global transcriptional activity of M. tuberculosis when exposed to the lysosomal soluble fraction (SF) pre-
pared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation con-
ditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory
conditions that led to the absence of apparent mycobacterial replication, M. tuberculosis expressed a unique transcriptome with
modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy re-
sponse, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of
increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific
responses, we identified the glgE-mediated 1,4 ␣-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin sys-
tems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A
meta-analysis including previously reported transcriptomic studies in macrophage infection and in vitro stress models was con-
ducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding gene Rv1258c was selected
for validation. An M. tuberculosis ⌬Rv1258c mutant was constructed and displayed increased susceptibility to killing by lyso-
somal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macro-
phage cell line THP-1.

M ycobacterium tuberculosis infects a third of the world’s pop-

ulation and causes death to millions of infected individuals
annually. While 90% of the infected population is able to prevent
protracted event, as evidenced by the detection of low numbers of
viable bacilli 7 days postinfection (7, 8). With an increasing num-
ber of mycobacterial factors reported to be specifically implicated
progression into active disease, incomplete sterilization of the in- in M. tuberculosis survival within activated macrophages (9), it
fecting bacilli, typically within granulomatous lesions formed in seems that this pathogen has evolved strategies to adapt and sur-
the lungs, leads to latent tuberculosis (TB), the asymptomatic vive within this hostile compartment, thereby challenging the idea
form of the disease. It is a longstanding paradigm that these lesions that the lysosomal compartment is a dead end for M. tuberculosis.
provide a niche environment that induces TB latency, where the Specific M. tuberculosis responses to the lysosomal environment
bacterium is believed to enter a state of bacteriostasis or very slow could therefore be exploited to identify novel targets and develop
replication with low energetic and metabolic activities and retains
the ability to resume growth under permissive conditions, leading
to disease reactivation (1).
Macrophages represent a large proportion of the cell popula- Received 24 January 2016 Returned for modification 12 February 2016
Accepted 10 June 2016
tions that are present in a TB lung granuloma (2, 3). Their phago-
Accepted manuscript posted online 20 June 2016
cytic abilities are responsible for eliminating most intracellular
Citation Lin W, de Sessions PF, Teoh GHK, Mohamed ANN, Zhu YO, Koh VHQ, Ang
microbes, and as such macrophages are important players in host MLT, Dedon PC, Hibberd ML, Alonso S. 2016. Transcriptional profiling of
innate immunity (4, 5). However, upon phagocytosis, internal- Mycobacterium tuberculosis exposed to in vitro lysosomal stress. Infect Immun
ized M. tuberculosis is able to survive and replicate within the 84:2505–2523. doi:10.1128/IAI.00072-16.
phagosome by blocking its fusion with lysosomes according to a Editor: S. Ehrt, Weill Cornell Medical College
process that involves several mycobacterial lipid and protein fac- Address correspondence to Sylvie Alonso, [email protected].
tors (6). Following the onset of cell-mediated immunity, however, * Present address: Wenwei Lin, Singapore Programme of Research Investigating
macrophage activation overrides phagosome maturation arrest New Approaches to Treatment of Tuberculosis (SPRINT-TB), Yong Loo Lin School
of Medicine, National University of Singapore (NUS), Singapore; Michelle Lay Teng
and delivers M. tuberculosis into the lysosomal compartment (7, Ang, Lee Kong Chian School of Medicine and School of Biological Sciences,
8), characterized by an increased acidic environment and contain- Nanyang Technological University, Singapore.
ing a plethora of bactericidal molecules, including hydrolytic en- Supplemental material for this article may be found at http://dx.doi.org/10.1128
zymes, oxygenated lipids, fatty acids, reactive oxygen species and /IAI.00072-16.
nitrogen intermediates, and antimicrobial peptides. However, Copyright © 2016, American Society for Microbiology. All Rights Reserved.
killing of mycobacteria in activated macrophages appears to be a

September 2016 Volume 84 Number 9 Infection and Immunity iai.asm.org 2505

Lin et al.

novel anti-TB drugs. However, there is limited knowledge on the mycin and kanamycin were added at 80 and 20 ␮g/ml, respectively. Hy-
behavior and physiology of M. tuberculosis in the lysosomal com- gromycin was purchased from Roche. Kanamycin, streptomycin, and car-
partment. bonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from
Transcriptional profiling of M. tuberculosis from infected mac- Sigma. For CFU enumeration, serial dilutions were performed in the
rophages of human or mouse origin has been the typical approach Middlebrook 7H9 medium and plated on Middlebrook 7H11 agar. Plates
were incubated at 37°C for 3 to 4 weeks.
to decipher the behavior of intramacrophage M. tuberculosis (10–
Determination of the MIC of streptomycin. Mid-log-phase myco-
15). A comparative study between resting and gamma interferon
bacterial cultures were grown in 7H9 medium and diluted to an optical
(IFN-␥)-activated macrophages identified a specific subset of my- density at 600 nm (OD600) of 0.02. The diluted bacterial suspension (200
cobacterial genes that were distinctly modulated in activated mac- ␮l) was added to 2-fold serially diluted streptomycin (5 ␮l) in a flat-
rophages, thereby supporting a lysosome-specific transcriptional bottom 96-well plate and incubated for 5 days. The OD600 of the cultures
reprogramming in M. tuberculosis with the potential to adapt to were measured using a Bio-Rad iMark microplate absorbance reader at
the inhospitable lysosomal microenvironment (11). While these 600 nm. The values were plotted against the log concentrations of strep-
studies have captured dynamic global transcriptional changes in tomycin, and a sigmoidal dose-response curve was fitted to the plot. The
M. tuberculosis during macrophage infection, contradictory ob- MIC corresponded to the concentration which inhibits 100% of visible
servations were also reported, likely due to underlying experimen- bacterial growth based on the OD600.
tal differences between these macrophage infection models, for Extraction of lysosomal SF. The lysosomal soluble fractions (SF) were
instance, the macrophage type and M. tuberculosis strains em- extracted from activated bone marrow-derived macrophages (BMMOs)
as previously described (27). T75 flasks of confluent BMMOs were incu-
ployed and/or the time postinfection at which the transcriptome
bated for 2 h at 37°C and 5% CO2 with 5 ml of 40 mg/ml iron-dextran (40
was assessed. The unsynchronized infection process throughout
kDa) mixed with 2⫻ Opti-MEM (Gibco) at a 1:1 ratio. The monolayers
the macrophage population could generate a transcriptional pro- were rinsed twice in 10 ml of warmed sterile phosphate-buffered saline
file representative of a combination of M. tuberculosis gene re- (PBS) to remove the excess Fe-dextran and chased overnight in culture
sponses to multiple microenvironments encountered during medium. The cells were scraped in 5 ml of homogenization buffer (HB)
macrophage infection which prevent the dissection of responses (250 mM sucrose, 0.5 mM EGTA, 0.1% gelatin, and 20 mM Tris, pH 7.0),
pertaining to each of the subcellular environmental niches en- centrifuged at 1,500 rpm at 4°C for 10 min, and lysed by passing through
countered by M. tuberculosis during its intramacrophage life. To a tuberculin syringe. The lysate was subjected to low-speed centrifugation
address these limitations, gene expression studies have been con- at 1,000 rpm at 4°C for 10 min to remove debris, nuclei, and intact cells.
ducted in defined in vitro culture settings that feature one partic- The supernatant was applied to a MiniMACS column (Miltenyi Biotech)
ular stress or growth condition possibly encountered by M. tuber- placed on a magnetic stand to retain the iron-loaded lysosomes. After two
culosis during macrophage infection, including hypoxia (16), washes with HB, the column was removed from the magnetic stand, and
the bound iron-loaded lysosomes were eluted twice with 500 ␮l of HB.
nitric oxide (17, 18), iron limitation (19), acidic pH (20), gradual
The lysosomes were spun down at 12,000 rpm for 30 min and stored as a
oxygen depletion (21, 22), nutrient starvation (23, 24), antibiotic
dry pellet at ⫺20°C until use. To prepare the lysosomal SF, each pellet
pressure (25), and stationary phase (26). These studies have al- was resuspended in 200 ␮l of SF buffer (1% Tween 20, 20 mM sodium
lowed the identification of M. tuberculosis genes that respond spe- acetate, pH 5.5). The lysates from eight pellets (2.5 ⫻ 108 cells) were
cifically to a particular environmental cue or growth condition. pooled and applied to two MidiMACS (Miltenyi Biotech) columns to
Our work aims to study the transcriptional response of M. remove iron. The flowthrough was collected and centrifuged at
tuberculosis to the lysosomal content using RNA sequencing 100,000 rpm at 4°C for 50 min. The supernatant corresponding to the
(RNA-seq). M. tuberculosis was exposed to the lysosomal soluble SF was collected and the total protein content was estimated using the
fraction (SF) prepared from activated macrophages. Previous bicinchoninic acid (BCA) protein assay kit (Thermo-Scientific Pierce).
work has shown that the lysosomal SF possesses mycobactericidal SF was stored at ⫺80°C until use.
activity in a dose- and time-dependent manner (27). Here, upon Bactericidal assays. Bactericidal assays on M. tuberculosis strains were
exposure to SF conditions that led to an absence of apparent my- performed with SF and synthetic human cathelicidin (LL-37; Peptide In-
stitute, Japan). LL-37 was reconstituted in 0.01% acetic acid for storage
cobacterial replication, we report a unique transcriptional signa-
in ⫺80°C until use. When required, 1 ␮g/ml CCCP was added to the
ture as part of M. tuberculosis adaptive response to the hostile medium. Mid-log-phase mycobacterial cultures of 5 ⫻ 105 CFU/ml were
lysosomal environment. treated with the indicated concentrations of the bactericidal agents for the
indicated periods of time. The number of surviving bacteria was enumer-
MATERIALS AND METHODS ated by plating appropriate dilutions of the mixture on 7H11 agar and
Ethics statement. All of the animal experiments were carried out under incubating at 37°C for 3 weeks.
the guidelines of the National Advisory Committee for Laboratory Ani- RNA isolation and qualitative real-time PCR. Mycobacterial cultures
mal Research (NACLAR) in the AAALAC-accredited NUS animal facili- were incubated with RNAprotect bacterial reagent (Qiagen) for RNA sta-
ties (http://nus.edu.sg/iacuc/). NUS has obtained a license (VR008) from bilization. The pelleted bacteria were then resuspended in 100 ␮l Tris-
the governing body Agri-Food & Veterinary Authority of Singapore EDTA (TE) containing 20 ␮g/ml lysozyme and incubated at room tem-
(AVA) to operate an Animal Research Facility. The animal experiments perature for 20 min. RNA extraction was then performed using an RNeasy
described in this work were approved by the IACUC from the National minikit (Qiagen) according to the manufacturer’s instructions. Contam-
University of Singapore under protocol number R2014-00723. inating genomic DNA from the eluted total RNA was removed using the
Bacterial strains and growth conditions. The parental strain of M. Turbo DNA-free kit according to the manufacturer’s protocol. The RNA
tuberculosis CDC1551, its derived mutant, and complemented strains concentrations and purity were measured using a NanoDrop 1000 spec-
were grown in Middlebrook 7H9 medium (Difco) supplemented with trophotometer (Thermo Scientific). Reverse transcription was performed
10% ADS [50 g bovine fraction V albumin, 20 g D-(⫹)-glucose, 8.1 g on 10 ng bacterial RNA using the iScript cDNA synthesis kit (Bio-Rad).
sodium chloride per liter], 0.05% Tween 80, and 0.5% glycerol or on Real-time PCR was performed in a 96-well plate with each well containing
Middlebrook 7H11 agar containing oleic acid-albumin-dextrose-catalase 2 ␮l cDNA mix, 0.5 ␮l forward (F) and reverse (R) primers (0.5 ␮M final),
(OADC; Becton Dickinson) and 0.5% glycerol. When appropriate, hygro- and 25 ␮l SYBR green supermix with ROX (Bio-Rad) to a final volume of

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 M. tuberculosis Gene Response to Lysosomal Stress

50 ␮l. The list of primers is presented in Table S7 in the supplemental pYUB854 plasmid at its corresponding multiple cloning sites (MCS)
material. Samples were run in triplicate. Real-time PCR amplification was flanking the hygromycin resistance gene, hyg. A PacI-restricted fragment
conducted with the ABI Prism 7500 sequence detector (Applied Biosys- containing the selection genes lacZ and sacB was obtained from pGOAL17
tems) over 40 cycles and with an annealing temperature of 61°C. The (32) and cloned into the pYUB854-PCR5=-3= construct to obtain the final
expression of each target gene was based on relative quantification (RQ) delivery vector, pYUB854-Rv1258c. The overexpressing complemented
using the comparative critical threshold (CT) value method. Relative strain Ox-Rv1258c was constructed by introducing Rv1258c, under the
quantification of a specific gene was evaluated in each reaction by normal- strong constitutive mycobacterial hsp60 promoter, into the ⌬Rv1258c
ization to the CT value obtained for the endogenous control gene, sigA. mutant strain using a promoter-less integrative plasmid, pMV306 (33).
For validation of transcriptome sequencing (RNA-seq) data, fold changes The mycobacterial hsp60 promoter was excised from pMV262 and cloned
(RQ values) were derived with reference to expression levels from M. into MCS of pMV306 vector. The ORF of Rv1258c was amplified from M.
tuberculosis incubated with SF buffer for 48 h. For validation of Rv1258c tuberculosis CDC1551 parental genomic DNA using primers indicated in
overexpression, the fold change was derived with reference to expression Table S7 and subsequently were inserted downstream of the hsp60 pro-
from WT M. tuberculosis. moter to obtain the final delivery vector, pMV306-Rv1258c. The UV-
RNA-seq library preparation. Total DNA-free RNA sample was de- irradiated plasmid solutions (1 ␮g) were electroporated into the respec-
pleted of bacteria rRNA with Ambion’s MICROBExpress kit (AM1905) tive M. tuberculosis strains as described previously (32). To identify the
per the manufacturer’s instructions. Bacterial rRNA-depleted sample was ⌬Rv1258c mutant, hygromycin-resistant white colonies were selected.
processed using the TruSeq RNA sample preparation (v2) per the manu- Deletion at the Rv1258c locus was verified by PCR using primers listed in
facturer’s instructions. Library preparation entailed fragmentation, 1st- Table S7 and Southern blot analysis. To identify the Ox-Rv1258c strain,
and 2nd-strand cDNA synthesis, end repair, A tailing, and ligation of kanamycin-resistant colonies were selected. Quantitative reverse tran-
adapters with multiplex indexes according to the manufacturer’s instruc- scription-PCR (qRT-PCR) was used to detect increased transcriptional
tions. Samples were enriched with 15 PCR cycles followed by Agencourt activity of Rv1258c.
AMPure XP magnetic bead (Beckman Coulter, Brea, CA, USA) clean up Southern blot analysis. Chromosomal DNA (1 ␮g) prepared from
according to the manufacturer’s instructions. The quality of cDNA librar- each M. tuberculosis strain was digested with EcoRI and XmaI for 4 h and
ies was checked with Agilent DNA1000 chips (2100 Bioanalyzer; Agilent subjected to 0.8% agarose gel electrophoresis. The agarose gel containing
Technologies, Santa Clara, CA, USA). Next-generation sequencing was the digested DNA was chemically treated and transferred onto a nitrocel-
performed using an Illumina HiSeq 2000 flow cell with two 76-bp end lulose membrane (Millipore) according to Roche’s digoxigenin (DIG)
runs. PhiX was used as a control. application manual. The membrane was UV fixed for 1 min and equili-
RNA-seq data analysis. RNA-seq data analysis was performed on the brated with 10 ml preheated DIG Easy Hyb solution (Roche) at 65°C for
CLC Genomics platform. Sequence reads were aligned to the Mycobacte- 20 min, with gentle agitation. A DIG-labeled probe was amplified using
rium tuberculosis CDC1551 parental reference genome (GenBank acces- the PCR DIG probe synthesis kit (Roche) according to the manufacturer’s
sion number NC_002755). The reads per kilobase per million (RPKM) instructions and primers as listed in Table S7. For hybridization, about 5
value for each gene was generated. Differential gene expression analysis to 25 ng/ml heat-denatured DIG-labeled DNA probe in DIG Easy Hyb
using the R edgeR package was performed for the following groups of data solution was incubated with the membrane overnight at 65°C. Detection
sets: incubation for 24 h or 48 h at 0, 10, or 20 ␮g/ml SF. The exact-test was performed using alkaline phosphatase (AP)-conjugated anti-DIG
function was applied to determine the association of the differences in antibody (Roche) at a dilution of 1:5,000. The membrane was devel-
expression read counts within each group, and corresponding P values oped using nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphos-
were adjusted using the default Benjamini & Hochberg procedure. Their phate (NBT-BCIP)-AP substrate (Chemicon).
adjusted P values, in ⫺log10 scale on the y axis and fold changes in log2 Macrophage survival assays. Bone marrow cells were flushed from
scale on the x axis, were plotted as a volcano plot. Differential gene expres- femurs of 6- to 8-week-old BALB/c mice, seeded onto petri dishes (4
sion was determined by a false discovery rate (FDR) of ⬍0.01. Genes with femurs per dish; Greiner), and differentiated into macrophages over 6
read counts of less than 5 from both SF-treated and nontreated groups days in BMMO complete medium supplemented with 10 ng/ml recom-
were also eliminated. Further functional annotation clustering analysis binant mouse macrophage colony-stimulating factor (rM-CSF; R&D Sys-
was performed using Database for Annotation, Visualization and Inte- tems). Differentiated macrophages were recovered by dislodging them in
grated Discovery (DAVID), version 6.7 (28, 29), and TB Database (http: cold 1⫻ PBS containing 1 mM EDTA (pH 7.4) and washed once in 1⫻
//www.tbdb.org/). PBS. To prepare activated macrophages, the complete medium was sup-
Meta-analysis with in vitro and ex vivo models of M. tuberculosis. plemented with 10% horse serum (Gibco) and macrophages were acti-
Microarray-based transcriptome studies of M. tuberculosis in in vitro and vated with 100 U/ml recombinant mouse IFN-␥ (Chemicon) and 50
ex vivo models of M. tuberculosis were selected for comparative analysis ng/ml of tumor necrosis factor (TNF) for 48 h. Primary macrophages
with the M. tuberculosis transcriptome generated in this study. For short- consistently represented 70 to 80% of the total cell population harvested,
term primary murine macrophage (BMMO) (10, 11) and human macro- as determined by flow cytometry using a panmacrophage marker, anti-
phage (THP-1) infection studies (13), genes that were differentially ex- F4/80 antibody (eBioscience). A human THP-1 monocytoid cell line
pressed at 24 h or 48 h after infection were considered. For temporal (ATCC TIB-202; ATCC, MD, USA) was maintained at 37°C and 5% CO2
studies based on BMMO (30), genes that exhibited significant temporal in HEPES buffered RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA)
trends were considered. For analysis with in vitro models of M. tuberculosis medium with 10% heat-inactivated fetal bovine serum (FBS), 2 mM
persistence, differential M. tuberculosis transcriptomes generated from L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4,500 mg/liter glu-
gradual hypoxic (21), defined hypoxic (22), nutrient starvation (24) and cose, and 1,500 mg/liter sodium bicarbonate (pH 7). When needed, cells
drug-tolerant persister (25) models were considered. were expanded into 75-cm2 flasks and were activated with retinoic acid
Construction of ⌬Rv1258c mutant and Ox-Rv1258c complemented (RA; 1 ␮M) and vitamin D3 (VD; 1 ␮M) for 3 days as described previously
strains. The ⌬Rv1258c mutant strain was generated in the M. tuberculosis (34). For survival assays, BMMO monolayers (5 ⫻ 104 cells/well) or
CDC1551 background by allelic exchange using the suicide plasmid back- RAVD-activated THP-1 cells (2.5 ⫻ 104 cells/well) in 24-well tissue cul-
bone pYUB854, as previously described (31). Briefly, primers with rele- ture plates (Nunc) were incubated with mycobacteria at multiplicities of
vant restriction enzyme sites (see Table S7 in the supplemental material) infection (MOI) of 2 and 5, respectively, for 45 min in their respective
were designed to amplify 5= and 3= PCR fragments (⬃1 kb) flanking the incomplete culture media (culture media without penicillin-streptomy-
Rv1258c open reading frame (ORF) from genomic DNA of the M. tuber- cin and FBS). Infected cells were washed twice with 1⫻ PBS, and the
culosis CDC1551 parental strain. The fragments were cloned into the respective complete culture medium without penicillin-streptomycin was

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Lin et al.

FIG 1 Mycobactericidal activity of a lysosomal soluble fraction (SF) prepared from activated primary murine macrophages. Mid-log-phase in vitro cultures of
M. tuberculosis CDC1551 strain were coincubated for 24 or 48 h with a lysosomal SF prepared from activated primary murine macrophages at the indicated
concentrations or with SF buffer only. (A) The treated bacteria were then plated on 7H11 agar and enumerated for viable CFU after 16 days of incubation at 37°C.
The dotted line represents the initial inoculum. (B) Results are expressed as a percentage of viable CFU obtained with buffer only at their respective times of
incubation. Data shown are the means ⫾ standard deviations (SD) from triplicates.

added to each well. At the indicated time points, cells were washed with led to an apparent replication arrest, which can be the result of (i)
1⫻ PBS and lysed with 0.1% Triton X-100 (Sigma-Aldrich) to release the a true arrest in replication where mycobacteria cease dividing but
intracellular bacteria. The cell lysates were serially diluted in 7H9 medium do not die, as described for other stress conditions, such as hyp-
and plated on 7H11 agar. The number of CFU was enumerated after oxia (35) or starvation (36), or (ii) equal killing and replication
incubation at 37°C for 16 days. rates that cancel each other out. Finally, the cidal condition was
Statistical analysis. Statistical significance was assessed by the Student
observed when M. tuberculosis was incubated with 30 ␮g/ml SF for
t test, and two-tailed P values of less than 0.05 were considered statistically
significant. 48 h, which resulted in a drastic reduction in viable CFU com-
Accession number. The data discussed in this publication have been pared to the 48-h control (Fig. 1B).
deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible RNA sequencing of M. tuberculosis in the LivE model. To
through GEO series accession number GSE68337. investigate the transcriptome profile of M. tuberculosis upon ex-
posure to SF, M. tuberculosis was exposed to noninhibitory (10
RESULTS AND DISCUSSION ␮g/ml SF, 24 h), subinhibitory (10 ␮g/ml SF, 48 h), and inhibitory
The LivE model. The lysosomal in vitro exposure (LivE) model (20 ␮g/ml SF, 48 h) conditions, with buffer only (0 ␮g/ml SF 24 h
consists of the direct exposure of M. tuberculosis to the soluble and 48 h) as the reference control. Illumina sequencing was per-
fraction of lysosomes (SF) purified from activated murine bone formed on biological triplicates of cDNA libraries prepared from
marrow-derived macrophages (BMMO) based on a previously mRNA extracted from the SF-treated M. tuberculosis cultures.
described protocol (27). The mycobactericidal activity of SF prep- High-quality paired-end sequence reads were generated for each
arations was determined by incubating mid-log-phase M. tuber- sample and were aligned with the M. tuberculosis CDC1551 paren-
culosis cultures with a range of SF total protein concentrations for tal reference genome, revealing coverage of more than 264 for all
24 and 48 h. As previously reported (27), the mycobactericidal samples and indicating a high accuracy in the sequences gener-
activity of SF was found to be both time and concentration depen- ated. More than 89% of the sequence tags were mapped to the
dent (Fig. 1A). M. tuberculosis remained viable and unaffected in annotated CDS in the sense orientation. Differential expression
its growth rate after 24 h of incubation within the range of SF analysis was performed with the R edgeR package (see Materials
concentrations tested, as evidenced by CFU values being compa- and Methods). We observed that the number of differentially ex-
rable to those of the positive control (buffer only) at 24 h. In pressed M. tuberculosis genes increased with increasing growth-
contrast, a significant and dose-dependent reduction in viable inhibitory SF conditions, with more genes being induced than
CFU was observed after 48 h of coincubation. repressed, as illustrated in the volcano plots (Fig. 2). In addition,
Based on the growth profiles observed, we defined the follow- the majority of the genes found to be modulated under noninhibi-
ing LivE conditions, ranging from noninhibitory to cidal upon SF tory conditions were further modulated under the subinhibitory
exposure. Noninhibitory conditions consist of exposing M. tuber- and inhibitory conditions.
culosis to 10 to 30 ␮g/ml SF for 24 h, which led to growth compa- The inhibitory LivE condition (iLivE) of 20 ␮g/ml for 48 h was
rable to 24 h of incubation with buffer only (24 h control) (Fig. 1). then selected for further analysis, where the apparent replication
The subinhibitory condition was achieved by exposing M. tuber- appears to resemble the nonreplicative state described for myco-
culosis to 10 ␮g/ml SF for 48 h and was characterized by a signifi- bacteria exposed to other environmental stresses, such as hypoxia
cant decrease in cell viability compared to the 48-h buffer control (35) or nutrient starvation (36) (Fig. 1). The iLivE M. tuberculosis
(Fig. 1) but a greater number of viable CFU compared to the 24-h genes were short-listed based on an FDR of ⬍0.01 and disregarding
control (Fig. 1A). The inhibitory condition was obtained upon genes with expression read counts of ⬍5 (see Table S1 in the supple-
incubation of M. tuberculosis in the presence of 20 ␮g/ml SF for 48 mental material). Gene function annotation was performed using
h, which resulted in a concentration of viable bacteria that was DAVID and TBDB databases. The distribution of iLivE M. tubercu-
comparable to the inoculum concentration and significantly losis genes into different functional categories showed that a sig-
lower than that obtained with the 48-h untreated control (Fig. 1A nificant number of genes were involved in cell wall remodeling
and B). This suggested that incubation with 20 ␮g/ml SF for 48 h and substrate transport, intermediary metabolism and respira-

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 M. tuberculosis Gene Response to Lysosomal Stress

TABLE 1 Functional categories representing the iLivE M. tuberculosis

transcriptome
No. of genesb
Functional categorya Induced Repressed
Cell wall and cell processes 27 15
Information pathways 17 2
Insertion sequences and phages 10
Intermediary metabolism and respiration 41 20
Metabolism 13 13
PE/PPE 5 7
Pseudogenesc 8
Regulatory proteins 15 2
Virulence, detoxification, and adaptation 29 11
Unknown 8 5
Conserved hypotheticals 91 31

Total 264 106

a
Functional annotations were obtained from TB Database (http://www.tbdb.org).
b
The number of induced and repressed genes is given for each gene category.
c
Genes that are not annotated in the M. tuberculosis H37Rv background.

tion, lipid metabolism, information pathways, regulatory pro-

teins, and virulence, detoxification, and adaptation (Table 1). In
total, there were 264 upregulated (Table 2) and 106 downregu-
lated (Table 3) iLivE M. tuberculosis genes.
Validation of RNA-seq data. To validate the gene expression
changes observed by RNA-seq analysis of the iLivE M. tuberculosis
transcriptome, we selected a number of genes that were either
highly modulated or of functional relevance and performed qRT-
PCR on M. tuberculosis exposed to the same iLivE conditions (20
␮g/ml for 48 h). The icl gene was used as a negative control, given
its nonsignificant regulation under iLivE (fold change, 1.24; P
value of 0.16). A comparable trend in fold changes obtained with
both qRT-PCR and RNA-seq was observed for all selected genes
(Fig. 3), thereby validating the iLivE M. tuberculosis transcriptome
profile generated by RNA sequencing.
iLivE M. tuberculosis expresses a unique transcriptome. (i)
General stress responses. The hostile nature of the lysosomal
content undoubtedly imposes stress on M. tuberculosis. Under
iLivE conditions, we observed the induction of several markers of
general stress response, which includes a number of chaperone
protein-encoding genes (groEL1, groEL2, groES, grpE, dnaJ1,
dnaK, and hspR) that are involved in the folding and translocation
of polypeptides and DNA repair (dinF and dinX) (37) (Table 2).
These genes have also been reported to be upregulated during
BMMO infections (10, 11, 30) and in lungs from TB patients (38),
indicative of a stressful environment during infection partly con-
tributed by the lysosomal contents. Furthermore, upregulation of
the Clp proteases, particularly clpP1, clpP2, and clpC2, suggests
prevalent protein degradation in iLivE M. tuberculosis. This obser-
vation may indicate a homeostatic response to prevent toxic ac-
cumulation of misfolded and aggregated proteins generated un-
der stressed conditions (39). ClpP1 and ClpP2 have been reported

FIG 2 Volcano plots of M. tuberculosis genes in the LivE model. Transcrip-

tomes of M. tuberculosis exhibited differential expression under noninhibitory
(10 ␮g/ml SF, 24 h) (A), subinhibitory (10 ␮g/ml SF, 48 h) (B), and inhibitory
(20 ␮g/ml SF, 48 h) (C) conditions. A total of 4,293 M. tuberculosis CDC1551
genes were annotated.

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Lin et al.

TABLE 2 Genes induced in iLivE M. tuberculosis

Designation for
strain:
Functional categorya CDC1551b H37Rv Gene Description Fold change SD
Cell wall and cell processes MT0201 Rv0191 Sugar transporter family protein 1.93 0.2
MT0409 Rv0399c lpqK Putative lipoprotein 2.37 0.5
MT0623 Rv0593 mce2E (lprL) Mce family protein 1.81 0.2
MT0700 Rv0671 lpqP Lipoprotein 2.09 0.3
MT0961 Rv0934 pstS1 (phoS1) Periplasmic phosphate-binding lipoprotein 1.90 0.8
MT1013 Rv0985c mscL Large-conductance ion mechanosensitive channel 1.95 0.2
MT1297 Rv1258c tap Putative Tap-like membrane efflux pump 5.94 0.3
MT1503 Rv1456c Antibiotic transport membrane ABC transporter 1.82 0.5
MT1519 Rv1473 Probable macrolide transport ATP-binding protein ABC 1.89 0.4
transporter
MT1642 Rv1607 chaA Cation/proton antiporter 1.97 0.3
MT1926 Rv1877 Drug transporter 1.94 0.4
MT1973 Rv1922 Peptidase, putative 1.88 0.8
MT1997 Rv1946c lppG Possible lipoprotein 1.84 0.9
MT2016 Rv1964 yrbE3A Membrane protein 2.05 1.3
MT2031 Rv1979c Amino acid permease 2.03 0.5
MT2040 Rv1986 Putative amino acid transporter 2.08 0.9
MT2097 Rv2037c Conserved transmembrane protein 1.84 0.5
MT2100 Rv2040c Sugar ABC transporter, permease protein 1.87 0.7
MT2101 Rv2041c Sugar ABC transporter, sugar-binding protein 1.83 1.2
MT2334 Rv2273 Probable conserved transmembrane protein 2.00 1.2
MT2339 Rv2281 pitB Putative phosphate permease 3.59 1.5
MT2469 Rv2398c cysW Sulfate transport membrane protein ABC transporter 1.93 0.6
MT2471 Rv2400c sbp Sulfate ABC transporter substrate-binding protein 1.85 0.4
MT2598 Rv2522c Peptidase, M20/M25/M40 family 1.80 0.2
MT2901 Rv2835c ugpA Probable Sn-glycerol-3-phosphate transport integral 2.22 0.8
membrane protein ABC transporter
MT3080 Rv3000 Possible conserved transmembrane protein 3.11 1.4
MT3951 Rv3843c Probable conserved transmembrane protein 1.87 0.4

Information pathways MT0691 Rv0662c DNA-binding protein, CopG family, transcriptional repressor 2.08 1.0
MT0699 Rv0670 end AP endonuclease, family 2 2.01 0.5
MT1338 Rv1299 prfA Peptide chain release factor 1 2.01 0.3
MT1463 Rv1420 Probable excinuclease ABC (subunit C nuclease) 1.77 0.3
MT1589 Rv1537 dinX DNA polymerase IV DinX 2.09 0.5
MT2042 Rv1988 ermMT rRNA adenine N-6-methyltransferase, putative 2.62 0.4
MT2247 Rv2191 DNA polymerase III, epsilon subunit, putative 1.90 0.4
MT2488 Rv2415c comE operon protein 1, putative 2.50 0.7
MT2535 Rv2460c clpP2 ATP-dependent Clp protease proteolytic subunit 2 2.12 0.5
MT2536 Rv2461c clpP1 Probable ATP-dependent CLP protease proteolytic subunit 1 2.18 0.8
MT2741 Rv2667 clpC2 ATP-dependent protease ATP-binding subunit 2.10 0.8
MT2902 Rv2836c dinF DNA damage-inducible protein F, putative 1.92 0.6
MT2904 Rv2838c rbfA Ribosome-binding factor A 2.02 0.5
MT2905 Rv2839c infB Translation initiation factor IF-2 2.01 0.6
MT2942 Rv2874 dipZ Cytochrome c biogenesis protein 2.19 0.5
MT3686 Rv3580c cysS Cysteinyl-tRNA synthetase 1 2.62 0.2
MT3942 Rv3834c SERYL-tRNA synthetase 1.84 0.4

Insertion sequences and MT0850 Rv0829 IS1605=, transposase, truncation 2.08 2.4
phages MT0873 Rv0850 IS1606=, transposase 2.02 1.2
MT0948 Rv0921 IS1535, resolvase 2.13 1.0
MT2069 Rv2013 IS1607, transposase 2.67 0.8
MT2070 Rv2014 IS1607, transposase 2.11 0.2
MT2497 Rv2424c IS1558, transposase 2.48 1.1
MT2732 Rv2655c Possible PhiRv2 prophage protein 2.03 0.1
MT2735 Rv2646 Integrase 1.94 1.0
MT2953 Rv2885c IS1539, transposase 1.80 0.7
MT3573.3 Bacteriophage protein 1.85 0.2

Intermediary metabolism MT0098 Rv0089 Putative methyltransferase 3.50 1.4

and respiration MT0207 Rv0197 lpqS Molybdopterin oxidoreductase 1.80 0.7
MT0337 Rv0322 udgA UDP-glucose 6-dehydrogenase 1.91 0.7
MT0511 Rv0492c Oxidoreductase, GMC family 1.94 0.3
(Continued on following page)

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 M. tuberculosis Gene Response to Lysosomal Stress

TABLE 2 (Continued)
Designation for
strain:
Functional categorya CDC1551b H37Rv Gene Description Fold change SD
MT0560 Rv0536 galE3 NAD-dependent epimerase/dehydratase family protein 1.80 0.4
MT0777 Rv0753c mmsA Methylmalonate-semialdehyde dehydrogenase 2.24 1.2
MT0888 Rv0865 mog Probable molybdopterin biosynthesis protein 1.81 0.1
MT0916 Rv0892 Monooxygenase, flavin-binding family 1.84 0.6
MT1128 Rv1096 Polysaccharide deacetylase, putative 1.83 0.3
MT1295 Rv1256c cyp130 Probable cytochrome P450 1.79 0.5
MT1339 Rv1300 papM (hemK) N-methylase 1.91 0.3
MT1368 Rv1326c glgB 1,4-␣-Glucan branching enzyme 1.89 0.5
MT1369 Rv1327c glgE Glucanase 1.88 0.4
MT1424 Rv1380 pyrB Probable aspartate carbamoyltransferase 1.82 0.6
MT1511 Rv1464 csd Cysteine desulfurase 1.89 0.4
MT1512 Rv1465 Nitrogen fixation protein NifU-related protein 1.77 0.3
MT1636 Rv1600 hisC1 Probable histidinol-phosphate aminotransferase 1.77 0.3
MT1658 Rv1622c cydB Membrane cytochrome D ubiquinol oxidase subunit II 1.85 0.3
MT1659 Rv1623c cydA Membrane cytochrome D ubiquinol oxidase subunit I 1.98 0.3
MT1667 Rv1631 coaE Probable dephospho-CoA kinase 1.80 0.5
MT1690 Rv1652 argC Probable N-acetyl-gamma-glutamyl-phosphate reductase 1.80 0.9
MT1767 Rv1726 Oxidoreductase, FAD binding 1.91 0.7
MT1902 Rv1854c ndh-2 NADH dehydrogenase 1.76 0.6
MT1987 Rv1937 Ferredoxin reductase, electron transfer component, putative 1.78 0.7
MT2103 Rv2043c pncA Pyrazinamidase/nicotinamidas 1.90 0.5
MT2274 Rv2217 lipB Lipoate biosynthesis protein B 1.91 0.7
MT2336 Rv2276 cyp121 P450 heme-thiolate protein 2.25 1.0
MT2511 Rv2436 rbsK Ribokinase 1.84 0.9
MT2572 Rv2497c bkdA (pdhA) Probable branched-chain keto acid dehydrogenase E1 1.78 0.4
component, alpha subunit
MT2797 Rv2725c hflX GTP-binding protein 1.99 0.3
MT2965 Rv2897c Mg chelatase 4.77 0.7
MT2967 Rv2899c fdhD Formate dehydrogenase accessory protein 1.99 0.4
MT2968 Rv2900c fdhF Possible formate dehydrogenase H 2.09 0.2
MT3065 Rv2987c leuD 3-Isopropylmalate dehydratase small subunit 1.86 0.2
MT3066 Rv2988c leuC 3-Isopropylmalate dehydratase large subunit 2.10 0.4
MT3224 Rv3137 Inositol monophosphatase family protein 1.95 0.7
MT3283 Rv3192 Putative luciferase 2.52 1.2
MT3514 Rv3406 Putative dioxygenase 2.46 0.7
MT3687 Rv3581c ispF Probable 2C-methyl-d-erythritol 2,4-cyclodiphosphate 1.82 0.2
synthase
MT3813 Rv3710 leuA 2-Isopropylmalate synthase 1.77 0.4
MT3950 Rv3842c glpQ1 Glycerophosphoryl diester phosphodiesterase 2.43 0.5

Metabolism MT0590 Rv0564c gpsA (gpdA1) Probable glycerol-3-phosphate dehydrogenase 2.06 0.2
MT0776 Rv0752c fadE9 Acyl-CoA dehydrogenase 2.43 1.1
MT1162 Rv1130 prpD Possible methylcitrate dehydratase 3.62 0.3
MT1163 Rv1131 gltA1 (prpC) Probable methylcitrate synthase 3.70 0.3
MT1518 Rv1472 echA12 Possible enoyl-CoA hydratase 1.80 0.6
MT1983 Rv1933c fadE18 Acyl-CoA dehydrogenase, putative 1.91 0.4
MT1984 Rv1934c fadE17 Acyl-CoA dehydrogenase 3.09 1.5
MT2243 Rv2188c pimB Mannosyltransferase 1.96 0.2
MT2599 Rv2523c acpS Holo-[acyl-carrier protein] synthase 1.90 0.3
MT2730 Rv2953 Enoyl reductase, may be involved in phenolpthiocerol and 2.18 1.4
phthiocerol dimycocerosate (dim) biosynthesis
MT3081 Rv3001c ilvC Ketol-acid reductoisomerase 1.98 0.3
MT3082 Rv3002c ilvH (ilvN) Acetolactate synthase, small unit 2.31 0.4
MT3083 Rv3003c ilvB Acetolactate synthase, large unit 2.37 0.5

PE/PPE MT0369 Rv0354c ppe7 PPE family protein 1.86 0.4

MT0778 Rv0754 PE_PGRS11 2.41 0.6
MT2505 Rv2430c ppe41 PPE41 1.77 0.9
MT3637 Rv3533c PPE62 2.10 0.0
MT3701 Rv3595c pe_pgrs59 PE/PGRS protein 1.85 0.2

Regulatory proteins MT0222 Rv0212c nadR AsnC family transcriptional regulator 4.02 0.6
MT0368 Rv0353 hspR Probable heat shock protein transcriptional repressor 2.22 0.5
MT0481 Rv0465c Transcriptional regulator 2.24 0.9
(Continued on following page)

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Lin et al.

TABLE 2 (Continued)
Designation for
strain:
Functional categorya CDC1551b H37Rv Gene Description Fold change SD
MT0514 Rv0494 Transcriptional regulator, GntR family 1.97 0.1
MT0605 Rv0576 Transcriptional regulator, ArsR family 2.57 0.1
MT0849 Rv0827 kmtR Transcriptional regulator, ArsR family 1.93 0.7
MT1161 Rv1129c Probable transcriptional regulator protein 4.38 0.8
MT1440 Rv1395 Transcriptional regulator, AraC family 1.86 0.1
MT1520 Rv1473A Possible transcriptional regulatory protein 2.22 1.1
MT1960 Rv1909c furA Ferric uptake regulation protein 3.86 1.3
MT2039 Rv1985c Transcription regulator 2.60 0.1
MT2073 Rv2017 Transcriptional regulatory protein 2.75 1.9
MT2386 Rv2324 Transcriptional regulator, AsnC family 1.89 1.1
MT2980 Rv2912c Probable transcriptional regulatory protein, TetR family 2.19 0.4
MT3290.1 Rv3197A whiB7 Probable transcriptional regulatory protein, WhiB-like 3.56 0.6

Virulence, detoxification MT0134 Rv0126 treS Trehalose synthase, alpha-amylase family protein 1.92 0.4
and adaptation MT0254 Rv0240 vapC24 Possible toxin 2.04 0.5
MT0265 Rv0251c hsp Heat shock protein, HSP20 family 3.06 2.4
MT0289 Rv0277c vapC25 Possible toxin 2.25 0.6
MT0365 Rv0350 dnaK Probable chaperone protein 3.00 1.0
MT0366 Rv0351 grpE Probable GrpE protein 3.36 1.5
MT0367 Rv0352 dnaJ1 Chaperone protein 2.70 1.0
MT0397 Rv0384c clpB Endopeptidase ATP binding protein chain B, heat shock 1.77 0.4
protein F84.1
MT0456 Rv0440 groEL2 60-kDa chaperonin 2 2.78 1.3
MT0574 Rv0549c vapC3 Possible toxin 1.85 0.6
MT0575 Rv0550c vapB3 Possible antitoxin 2.23 0.6
MT0618 Rv0589 mce2A Mce family protein 1.76 0.2
MT0621 Rv0591 mce2C Mce family protein 2.56 0.1
MT0685 Rv0656c vapC6 Possible toxin 2.65 0.3
MT0693 Rv0665 vapC8 Possible toxin 2.02 2.7
MT1296 Rv1257c Probable oxidoreductase 2.27 0.2
MT1959 Rv1908c katG Catalase-peroxidase 2.34 0.2
MT1961 Rv1910c Hypothetical exported protein 2.09 0.1
MT2004 Rv1955 higB Possible toxin 2.72 0.9
MT2005 Rv1956 higA Possible antitoxin 2.05 0.6
MT2018 Rv1966 mce3A Mce family protein 2.05 0.4
MT2489 Rv2416c eis Enhanced intracellular survival protein 3.73 0.6
MT2503 Rv2428 ahpC Alkyl hydroperoxide reductase C protein 2.19 0.6
MT2504 Rv2429 ahpD Alkyl hydroperoxide reductase D protein 2.22 1.1
MT2941 NA Prevent-host-death family protein 3.00 3.8
MT3526 Rv3417c groEL1 60-kDa chaperonin 1 3.18 2.3
MT3527 Rv3418c groES 10-kDa chaperonin 2.79 1.6
MT3771 Rv3670 ephE Epoxide hydrolase 2.11 0.8
MT3949** Rv3841 bfrB Bacterioferritin 2.21 0.7
a
Conserved hypothetical, unknown, and pseudogenes are listed in Table S1 in the supplemental material.
b
**, DosR-dependent genes.

previously to play an important role in M. tuberculosis pathogen- tuberculosis (Fig. 2C), a key bifunctional enzyme that is induced
esis and represent potential drug targets (40). More recently, simultaneously with prpC and prpD in intracellular M. tuberculosis
ClpP1 has been used in a novel target mechanism-based whole- (30) to utilize fatty acids via the glyoxylate shunt and methylcitrate
cell screening assay and was used to successfully identify bort- cycle (44). The sole induction of icl has been reported in the pres-
ezomib as a new lead compound for tuberculosis therapy (41). ence of palmitic acid (11), suggesting that icl expression is directly
(ii) Metabolic reprogramming. Metabolic adaptations to host modulated by the presence of fatty acids, which may explain its
fatty acids and cholesterol by intracellular M. tuberculosis have lack of induction in the fatty acid-free iLivE model. Reinforcing
been reported in transcriptomics studies from macrophage and the notion that fatty acids are the preferred carbon source of in-
mouse infections (11, 13, 15, 30, 42). Upregulation of prpC and tracellular M. tuberculosis, we also found a number of iLivE M.
prpD was observed in iLivE M. tuberculosis (Table 2). These genes tuberculosis genes predicted with enzymatic functions involved in
encode key enzymes of the methylcitrate cycle and help M. tuber- biochemical activation and ␤-oxidation of fatty acids. These in-
culosis detoxify propionyl-coenzyme A (CoA), a product from clude acyl-CoA dehydrogenase (fadE9, fadE15, fadE17, and
fatty acid catabolism during intracellular survival (43). However, fadE18), fatty acid-CoA ligase (fadD5 and fadD9), enoyl-CoA hy-
induction of isocitrate lyase (icl) was not observed in iLivE M. dratase (echA12), and lipases (lipF and lipQ) (Table 2). Most of

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 M. tuberculosis Gene Response to Lysosomal Stress

TABLE 3 Genes repressed in iLivE M. tuberculosis

Designation in strain:
a
Functional category CDC1551b H37Rv Gene Description Fold change SD
Cell wall and cell MT0046 Rv0040c mtc28 Secreted proline-rich protein 0.53 0.3
processes MT0182 Rv0173 mce1E (lprK) Mce family protein 0.48 0.2
MT0356 Rv0341 iniB Isoniazid-inducible gene protein 0.47 0.2
MT0911 Rv0888 Probable exported protein 0.54 0.1
MT1235 Rv1197 esxK ESAT-6-like protein 0.34 0.1
MT1236 Rv1198 esxL ESAT-6-like protein 0.42 0.1
MT1729 Rv1690 lprJ Probable lipoprotein 0.43 0.1
MT1779 Rv1737c narK2 Nitrite extrusion protein, MFS 0.32 0.1
MT1932 Rv1884c rpfC Probable resuscitation-promoting factor 0.55 0.3
MT2411 Rv2346c esxQ ESAT-6-like protein 0.55 0.2
MT2412 Rv2347c esxP ESAT-6-like protein 0.53 0.2
MT2420 Rv2346c esxO ESAT-6-like protein 0.47 0.2
MT2458 Rv2389c rpfD Resuscitation-promoting factor 0.51 0.2
MT3988 Rv3874 esxB (cfp10) 10-kDa ESAT-6-like protein 0.56 0.2
MT3989 Rv3875 esxA (esat-6) ESAT-6-like protein 0.52 0.2

Information pathways MT2669 Rv2592c ruvB Holliday junction ATP-dependent DNA helicase 0.45 0.3
MT3347 Rv3249c Transcriptional regulator, TetR family 0.41 0.3

Intermediary metabolism MT0037 Rv0032 Aminotransferase 0.47 0.1

and respiration MT0266 Rv0252 nirB Probable nitrite reductase [NAD(P)H], large subunit 0.54 0.2
MT0738 Rv0711 atsA Possible arylsulfatase 0.51 0.3
MT1449 Rv1405c Methyltransferase 0.43 0.2
MT1603 Rv1552 frdA Probable fumarate reductase 0.51 0.0
MT1604 Rv1553 frdB Fumarate reductase, iron-sulfur subunit 0.42 0.0
MT1606 Rv1555 frdD Fumarate reductase membrane anchor subunit 0.42 0.1
MT1778** Rv1736c narX Probable nitrate reductase 0.51 0.9
MT1904 Rv1856c Possible oxidoreductase 0.54 0.2
MT2063** Rv2007c fdxA Ferredoxin 0.47 0.6
MT2088** Rv2029c pfkB 6-Phosphofructokinase 0.36 0.5
MT2401 Rv2338c moeW Possible molybdopterin biosynthesis protein 0.53 0.2
MT3194 Rv3111 moaC Molybdenum cofactor biosynthesis protein C 2 0.34 0.2
MT3423 Rv3322c Possible methyltransferase 0.37 0.1
MT3424 Rv3323c moaDE Molybdopterin cofactor biosynthesis protein D/E 0.37 0.2
MT3426 NA moaB3 Probable pterin-4-alpha-carbinolamine dehydratase 0.28 0.2
MT3427 NA moaA3 Molybdenum cofactor biosynthesis protein A 3 0.27 0.2
MT3849 Rv3741c Possible oxidoreductase 0.57 0.3
MT3850 Rv3742c Possible oxidoreductase 0.52 0.3
MT3969 Rv3854c ethA Monooxygenase 0.53 0.3

Metabolism MT0038 Rv0033 acpA (acpP) Acyl carrier protein 0.48 0.3
MT0175 Rv0166 fadD5 Probable fatty-acid-CoA ligase 0.55 0.1
MT0258 Rv1467c fadE15 Acyl-CoA dehydrogenase 0.45 0.2
MT1702 Rv1662 pks8 Probable polyketide synthase 0.55 0.2
MT2559 Rv2485c lipQ Carboxylesterase family protein 0.50 0.3
MT2667 Rv2590 fadD9 Fatty acid-CoA ligase 0.50 0.3
MT3216** Rv3130c tgs1 Triacylglycerol synthase 0.24 0.1
MT3326 Rv3229c desA3 Linoleoyl-CoA desaturase, putative 0.55 0.2
MT3348 Rv3250c rubB Rubredoxin 0.41 0.3
MT3349 Rv3251c rubA Rubredoxin 0.47 0.3
MT3350 Rv3252c alkB Transmembrane alkane 1-monooxygenase 0.44 0.3
MT3591 Rv3847c lipF Probable esterase/lipase 0.53 0.2
MT3933 Rv3825c pks2 Mycocerosic acid synthase 0.51 0.2

PE/PPE MT1233 Rv1195 pe13 PE family protein PE13 0.29 0.1

MT1234 Rv1196 ppe18 PPE family protein PPE18 0.33 0.0
MT1745 Rv1705c ppe22 PPE family protein PPE22 0.49 0.2
MT1746 Rv1706c ppe23 PPE family protein PPE23 0.47 0.1
MT2166 Rv2107 pe22 PE family protein PE22 0.42 0.1
(Continued on following page)

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Lin et al.

TABLE 3 (Continued)
Designation in strain:
a
Functional category CDC1551b H37Rv Gene Description Fold change SD
MT3427.1 Rv3347 ppe55 PPE family protein PPE55 0.27 0.2
MT3854 Rv3746c pe34 PE family protein 0.42 0.2

Regulatory proteins MT3230 Rv3143 Probable response regulator 0.53 0.2

MT3870 Rv3765c tcrX Probable two-component transcriptional regulatory 0.51 0.2
protein

Virulence, detoxification, MT0052 Rv0046c 1-l-myo-inositol-1-phosphate synthase 0.49 0.3

and adaptation MT0176 Rv0167 yrbE1A Conserved integral membrane protein 0.54 0.2
MT0178 Rv0169 mce1A Mce family protein 0.54 0.1
MT0179 Rv0170 mce1B Mce family protein 0.54 0.1
MT0180 Rv0171 mce1C Mce family protein 0.52 0.2
MT0181 Rv0172 mce1D Mce family protein 0.50 0.2
MT0183 Rv0174 mce1F Mce family protein 0.46 0.2
MT2087 NA Universal stress protein family protein 0.35 1.0
MT2090** Rv2031c hspX Alpha crystallin, 14-kDa antigen 0.25 0.0
MT2698** Rv2623 TB31.7 Universal stress protein family 0.43 0.5
MT3598 Rv3494c mce4F Mce family protein 0.50 0.1
a
Conserved hypothetical and unknown genes are listed in Table S1 in the supplemental material.
b
**, DosR-dependent genes.

these genes have been reported previously to be modulated under host environment (46). In line with the apparent nonreplicative
various in vitro and ex/in vivo conditions (10, 11, 21, 24, 30) with state of iLivE M. tuberculosis, we detected a downregulation of
the exception of fadE17 and fadE18, which seem to be specifically pks2 and desA3 (Table 3), which are essential genes for the biosyn-
upregulated in iLivE M. tuberculosis. Thus, it appears that the lys- thesis of main cell wall components of mycobacteria (47). On the
osomal content represents an environmental signal for the bacte- contrary, pks2 expression was found to be induced upon phago-
rium to upregulate genes involved in fatty acid beta-oxidation, some acidification (12, 20), an environmental cue that is not rep-
perhaps in anticipation of the next round of infection upon lysis of resented in the LivE model, where pH is maintained at 6.8. Inter-
the host cell. estingly, desA3 was previously proposed to be involved in
(iii) Cell wall remodeling. With a lipid-rich cell wall envelope, regulating the membrane fluidity necessary for physiological
mycobacterial cell wall remodeling is also tightly associated with function (48). Repressed expression of desA3 in iLivE M. tubercu-
its lipid metabolism (45) and can be a possible adaptive mecha- losis suggests reduced cell membrane fluidity, leading to limited
nism of M. tuberculosis when coping with a constantly changing barrier permeability that could limit drugs from gaining access to
their bacterial targets and consequently conferring phenotypic
drug resistance.
Furthermore, consistent with observations in the short-term
macrophage infection model (10, 11), the mce1 operon (yrbE1A
and mce1A-F) was downregulated in iLivE M. tuberculosis (Table
3). Deletion of this operon has been associated with (i) accumu-
lation of free mycolic acids in the mycobacterial cell wall (49), (ii)
a hypervirulent infection profile in mice with an impaired ability
to trigger a proinflammatory response (50), and (iii) in vitro phe-
notypic drug tolerance (51). Therefore, mce1 downregulation in
iLivE M. tuberculosis may lead to changes in its cell wall mycolic
acid composition, which could contribute to altered drug suscep-
tibility of M. tuberculosis.
Finally, in iLivE M. tuberculosis we measured the upregulation
of pstS1, which encodes a mycobacterial cell wall adhesin that has
FIG 3 Validation by quantitative real-time PCR analysis of selected iLivE M. been demonstrated to promote phagocytosis of mycobacteria via
tuberculosis genes. Exponential M. tuberculosis culture was coincubated with
SF under inhibitory conditions (20 ␮g/ml SF, 48 h) or with buffer only (con-
binding to the mannose receptor (52). This implies an increased
trol). Total RNA was extracted and real-time PCR was performed using spe- ability of bacilli to infect neighboring host cells when released
cific primers listed in Table S7 in the supplemental material. For each gene, the from apoptotic macrophages.
average from technical triplicates was calculated and expressed as fold change Downregulation of dosR-dependent genes. Induction of the
compared to the gene expression level measured in the control. Fold changes
(black bar) were compared to those obtained by RNA-seq (open bar). Data
transcriptional factor DosR, involved in activation of the dor-
shown are the means ⫾ standard deviations (SD) from triplicates. *, DosR- mancy program in M. tuberculosis (53), has been associated with
dependent genes. hypoxic conditions within TB granulomas (54) and reactive nitro-

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 M. tuberculosis Gene Response to Lysosomal Stress

gen intermediates produced by activated macrophages (55). The plemental material), suggests that these genes respond to multiple
Dos regulon, which comprises ⬃49 genes regulated by the DosR- environmental stimuli. Alternatively, intrinsic ROS production
S/T two-component system, has been described to respond to a by the mycobacterial cell itself could also be responsible for induc-
variety of signals and stresses, including low-oxygen tension, S- ing these genes. This hypothesis is supported by the upregulation
nitrosoglutathione (GSNO), ethanol, and carbon monoxide (56). of Rv1464 and Rv1465 ORFs (Table 2) from the SUF operon that
As expected, dosR expression was minimally modulated in the encodes the alternative mycobacterial iron-sulfur cluster machin-
iLivE model (fold change, ⫺1.3; P value of 0.25), since this model ery (64). Furthermore, high production of ROS was detected in
does not incorporate any of the above-mentioned stimuli. How- nonreplicating nutrient-starved mycobacteria and was attributed
ever, and interestingly, we observed significant modulation of 17 to cytochrome P450 (CYP)-based metabolism of ketone bodies
dosR-dependent genes in iLivE M. tuberculosis, among which 15 generated from triacylglycerol (TAG) stores during nutrient star-
were downregulated (Table 3). The majority of these repressed vation (our unpublished observations). Coincidentally, cyp121
dosR-dependent genes were also found downregulated in the and cyp130 were found to be upregulated in iLivE M. tuberculosis
long-term BMMO infection model (at day 8) (Table 3), and this (Table 2). Thus, intracellular production of ROS through CYP
was attributed to the sudden loss of cue(s) driving the DosR re- activity upon lysosomal exposure represents an interesting possi-
sponse at the later stage during macrophage infection (30), where bility which remains to be further investigated.
the bacterium presumably has transited to a persistent state. Con- Respiratory status. The respiratory status of M. tuberculosis is
sistent with this, transient and early induction of dosR-dependent dependent on the microenvironment it encounters, such as the
genes during the first 4 to 8 h, followed by a gradual decline to oxygen tension and availability of various carbon and nitrogen
baseline within 24 h, was also reported in a defined hypoxia model sources to act as terminal electron acceptors (65). Induction of
(22). bd-type terminal oxidase-encoding genes (cydA and cydB) and
Thus, these observations suggest that the gene expression sig- genes involved in nitrate respiration (narK2) were detected in
nature of iLivE M. tuberculosis partially overlaps that profiled at iLivE M. tuberculosis (Table 2). This is consistent with a transi-
the late stage of macrophage infection, which further supports the tional respiratory state previously described for intracellular M.
idea that mycobacteria at this stage of infection are exposed to a tuberculosis upon NO production following immune cell activa-
lysosomal environment. The data also suggest that genes previ- tion (65). In contrast, the fumarate reductase gene cluster frdABD
ously identified as part of the Dos regulon also are regulated inde- was notably repressed in iLivE M. tuberculosis, which is consistent
pendently of DosR. with the aerobic setup of the LivE model (Table 3). Induced ex-
ESAT-6 and PE-PPE family of proteins. Genes encoding a pression of fumarate reductase was observed in activated BMMOs
cluster of ESAT-6-like proteins (esat-6, cfp10, esxQ, esxP, esxK, (11) in an NO-dependent manner and in hypoxic lung lesions
and esxQ) were notably downregulated in iLivE M. tuberculosis from tuberculosis patients (38), and it was associated with anaer-
(Table 3). Consistent with this, the expression of esxQ, esxP, esxK, obic persistence (66). Interestingly, modulation of gene clusters
and esxQ genes was also found downregulated in resting and ac- encoding F0F1 ATP synthase (atpA-H) and NADH dehydrogenase
tivated macrophages (10). ESAT-6 and CFP-10 have been identi- 1 (nuoA-N), which are involved in aerobic respiration, was not
fied as both virulence factors and protective antigens (57, 58). In observed in iLivE M. tuberculosis. Along with ribosomal proteins
contrast, the PE/PPE genes that were modulated in iLivE M. tu- (rps), these regulons were also notably repressed during regulated
berculosis exhibited various expression trends in previous tran- slow growth (67) and in models of persistence where mycobacte-
scriptomics studies of macrophage (10, 11), mouse (42) infection rial replication arrest is induced by reduced oxygen and/or nutri-
models, and under in vitro stresses (21, 24) (see Table S1 in the ent availability (68, 69). As mentioned earlier, the true replicative/
supplemental material). Our analysis singled out PPE41 based on nonreplicative status of mycobacteria during SF exposure remains
its consistent induced profile observed in M. tuberculosis from to be further characterized.
infected BMMO (11) and human macrophages (13), mouse lungs Meta-analysis with transcriptomes of intracellular M. tuber-
(42), and iLivE M. tuberculosis (see Table S1). The PE25/PPE41 culosis from macrophage infection models. The experimental
protein complex was shown to induce dendritic cell activation and setup of the LivE model was designed to (partially) mimic expo-
drive Th2-biased immune responses (59), whereas Th1-biased sure of M. tuberculosis to the lysosomal content upon phagosome/
immune responses have long been known to be protective against autophagosome maturation during macrophage infection. Thus,
tuberculosis (60). Downregulation of ESAT-6-like proteins and we subjected the iLivE M. tuberculosis transcriptome profile to a
upregulation of PPE41 in iLivE M. tuberculosis indicates that the comparative analysis with selected key transcriptome profiling
lysosomal environment contributes to subversion of the host im- studies of intracellular M. tuberculosis during infection in BMMO
mune system by M. tuberculosis toward nonprotective immune and human macrophages (THP-1). With the caveat that each
responses. study used different infection conditions, different macrophage
Fighting oxidative stresses. Exposure to inhibitory SF condi- types, and different M. tuberculosis strains, our analysis revealed
tions upregulated several M. tuberculosis genes (furA, katG, ahpC, that 193 out of 370 iLivE genes overlapped genes from these mac-
and ahpD) (Table 2) that are known to be instrumental in com- rophage infection studies, while 177 did not overlap any of the
bating oxidative stresses mediated by reactive oxygen species studies considered (Fig. 4; see also Tables S2 and S3 in the supple-
(ROS) and reactive nitrogen intermediates (RNI) (61–63), which mental material). As expected, the largest overlap was observed
are abundantly produced in an activated macrophage. While furA with the BMMO infection models (168 out of 193) (Fig. 4; see also
and katG have been reported to be upregulated under in vitro Table S3), suggesting a partial recapitulation of intramacrophage
oxidative stresses (11), their regulation under nonoxidative con- lysosomal exposure in the LivE model. However, it is believed that
ditions, such as nutrient starvation (24), gradual hypoxia (21), in some of these BMMO infection models, mycobacteria reside
static growth (26), and iLivE conditions (see Table S1 in the sup- primarily in a phagosomal environment due to phagosome

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Lin et al.

and a primary mouse-derived macrophage (SF was prepared from

activated BMMO). Nevertheless, genes involved in oxidative
stresses (ahpC and ahpD) and fatty acid metabolism (prpC and
prpD) were found in the 25 iLivE gene subset that were commonly
regulated in both BMMO and THP-1 infection models (Fig. 4; see
also Table S3 in the supplemental material), indicative of a com-
parable intramacrophage environment between human and
mouse macrophages. From the subset of 11 iLivE genes that over-
lap THP-1 macrophages only (Fig. 4; see also Table S3), it is worth
mentioning rpfD, the product of which has been implicated in
resuscitating mycobacteria from dormancy (76).
Our meta-analysis also revealed a significant number of genes
FIG 4 Venn diagram comparing transcriptional profiles of M. tuberculosis
under iLivE conditions and during macrophage infections. THP-1, human
(177 iLivE M. tuberculosis genes) that did not overlap any of the
macrophage model; iLivE, inhibitory condition of lysosomal in vitro exposure. macrophage infection models analyzed (see Table S2 in the sup-
The asterisk indicates that the gene is included if it is modulated in at least one plemental material). These nonoverlapping genes could represent
of the three primary murine macrophage (BMMO) infection models. a subset of lysosome-inducible genes, which were previously not
detected in macrophage models due to nonsynchronized infec-
tion conditions, where only a small percentage of mycobacteria
maturation arrest. Therefore, it is possible that some of these harvested from the infected macrophages actually reside inside the
overlapping genes are also modulated by stimuli present in the lysosomal compartment at one time. Among these nonoverlap-
phagosome prior to lysosomal fusion. To further refine the lys- ping genes, we found glgE, glgB, and treS to be significantly
osome-specific gene responses triggered under iLivE conditions, upregulated (see Table S2). These genes are involved in the ␣-1,4-
we excluded iLivE genes that were modulated during resting mac- glucan pathway implicated in detoxification of maltose-1-phos-
rophage infection reported by Homolka et al. (10) and Schnap- phate (M1P) to ␣-glucan (77). Interestingly, loss of glgE, which
pinger et al. (11), where mycobacteria are believed to reside encodes a maltosyltransferase, was reported to impair the bacte-
mainly within phagosomes. Furthermore, since the process of in- rium’s replication ability in lungs and spleen from infected
fection is not synchronized, the bacilli retrieved from the infected BALB/c mice (77). This defect was attributed to cell toxicity from
macrophage population at each time point consist of a heteroge- the accumulation of the phosphosugar intermediate, thus reveal-
neous population of bacteria which have been exposed to various ing ␣-glucan synthesis as a potential target for antimicrobials (77).
intracellular environments, ranging from early endosome to lys- Upregulation of glgE in iLivE M. tuberculosis further indicates that
osomal compartment. Therefore, the mycobacterial transcrip- targeting of GlgE and the ␣-glucan synthesis pathway represents a
tional response measured is likely to be heterogeneous, reflecting viable therapeutic approach to kill intraphagolysosomal myco-
the responses to various intracellular microenvironments. On the bacteria.
contrary, in the iLivE model, the mycobacterial transcriptional Among the nonoverlapping iLivE gene subset, a number of
response is expected to be more homogeneous. Therefore, we pos- mycobacterial toxin/antitoxin (TA) systems were also signifi-
tulated that fold changes measured in iLivE M. tuberculosis are cantly induced. They include vapB3, vapC3, and vapC8 of the
likely to be greater in magnitude than those measured in activated VapBC systems, which represents the largest family of bacterial
macrophages. Thus, a more stringent cutoff value was arbitrarily TA systems (78). In addition, the HigBA1 TA system and three
implemented for each gene by either dividing (upregulation) or more VapC toxin-encoding genes, vapC24, vapC25, and vapC6,
multiplying (downregulation) their expression fold change by 1.5, were upregulated (Table 2). Upregulation of TA systems in re-
leading to selection of 41 iLivE M. tuberculosis genes (see Table sponse to adverse conditions has been described in other bacterial
S6). Among these 41 genes, most of the general stress response species (79). Typically, bacterial TA systems are made of a toxin
markers (groEL1, groES, and dnaJ1) were present, further support- protein and a more labile antagonistic antitoxin, which can be a
ing the increased level of stress experienced by bacilli exposed to protein or noncoding RNA (80). Some of the mycobacterial VapC
the lysosomal environment. Interestingly, we found several anti- toxins have been shown to exert a bacteriostatic effect on myco-
biotic resistance-related genes, namely, eis (Rv2416c), ermMT bacterial growth through their RNase activity (81). While nutri-
(Rv1988), and tap (Rv1258c), with remarkably increased expres- ent-starved M. tuberculosis cells and drug-tolerant M. tuberculosis
sion under iLivE conditions. Although the mechanisms by which persisters have been shown to express distinct sets of TA systems
they induce antibiotic resistance are different (70–73), expression (25, 68), the VapBC TA systems could be exploited to induce
of all of these genes is under the control of the transcriptional toxicity in intramacrophage bacilli.
factor WhiB7 (70), one of the earliest and most highly induced Finally, genes encoding cell wall-associated proteins, compris-
transcriptional regulators in M. tuberculosis during BMMO infec- ing transporters and lipoproteins, represent another prominent
tion (12), and in response to numerous stress conditions (70, 74, group of significantly induced nonoverlapping iLivE genes (see
75). Thus, these findings strongly support that the lysosomal con- Table S2 in the supplemental material). The transporters were
tent induces a WhiB7-mediated phenotypic drug resistance in M. mainly associated with drug efflux (Rv0191, Rv1877, and Rv1456c)
tuberculosis. and uptake of sulfate, amino acids, and sugars (cysW, Rv1979c,
In contrast, we observed a limited number of overlapping and pitB), while the lipoproteins exhibited a plethora of predicted
genes (36 out of 370) between THP-1 macrophage infection and functions, including peptidoglycan cross-linking and remodeling
the iLivE model (Fig. 4), which is likely attributable to inherent (lpqK and Rv1922), degradation processes (Rv0671), host cell ad-
physiological differences between a human macrophage cell line hesion and invasion (Rv0593), and as solute binding proteins of

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 M. tuberculosis Gene Response to Lysosomal Stress

macrophage or a granuloma, exposure to gradual hypoxia (21),

defined hypoxia (22), nutrient starvation (24), and antibiotics at
incomplete sterilizing concentrations (25) induces M. tuberculosis
persistence, characterized by nonreplication and phenotypic re-
sistance to the TB drugs isoniazid and rifampin. The meta-analysis
between iLivE M. tuberculosis transcriptome and M. tuberculosis
transcriptomes profiled from these in vitro models indicates min-
imal overlap, which suggests that the physiological state of myco-
bacteria exposed to lysosomal SF significantly differs from that of
hypoxic conditions, nutrient-starved conditions, or antibiotic-ex-
posed mycobacteria (see Tables S4 and S5 in the supplemental
FIG 5 Venn diagram comparing transcriptional profiles of M. tuberculosis material). Nevertheless, 115 iLivE genes overlapped at least one of
under iLivE conditions and various environmental stresses. EHR, defined hy- these in vitro models of persistence, with only four genes (echA12,
poxic model; persisters, drug-tolerant persisters model; starvation, nutrient hsp, higA, and Rv2036) commonly regulated in all models consid-
starvation model; NRP, gradual hypoxic model; iLivE, inhibitory condition of
lysosomal in vitro exposure. ered, excluding the drug persisters model (Fig. 5; see also Table
S5). The overlap between iLivE and at least one in vitro model here
suggests that the same pathway is triggered by more than one
ABC transport systems (sbp) (82). This could represent responses environmental stimulus, as supported by a substantial number of
to (i) membrane stresses induced by membrane-perturbing regulatory proteins present in this gene subset (see Table S5), in-
agents (antimicrobial cationic peptides present in the lysosomal cluding multistress-induced transcriptional factor WhiB7. On the
SF), (ii) intracellular toxic accumulation arising from SF-induced contrary, genes that are triggered by a specific environmental
metabolic changes, and/or (iii) membrane remodeling for nutri- stimulus, such as acidic pH, induced aprABC locus (83), and hy-
ent-scavenging activities or to facilitate escape into the cytosol. poxia-driven dosR, were not found in the iLivE transcriptome due
Meta-analysis with M. tuberculosis transcriptomes from in to the absence of these cues from the LivE model, highlighting a
vitro stress models. Under in vitro conditions that are meant to general limitation of in vitro models, where only one or a limited
reproduce some of the environmental cues encountered within a number of environmental stimuli are incorporated. On the other

FIG 6 Construction of M. tuberculosis ⌬Rv1258c mutant and its complemented strain, Ox-Rv1258c. (A) Schematic organization of CDC1551 M. tuberculosis
Rv1257c-1258c locus in parental strain (WT). The mutant was obtained by introducing a hyg cassette into the Rv1258c ORF by double homologous recombina-
tion and complemented by reintegrating Rv1258c under the M. tuberculosis hsp60 promoter (Ox-Rv1258c) into its genome. Southern blot probe and restriction
enzyme sites are indicated (E, EcoRI; X, XmaI; probe, double-headed arrow). (B) Southern blot analysis. M, DIG-labeled molecular ladder. (C) Transcriptional
activity of Rv1258c in WT, ⌬Rv1258c, and Ox-Rv1258c strains as determined by real-time PCR analysis. Data are expressed as averages ⫾ SD from triplicates. (D)
In vitro growth kinetics of WT and ⌬rv1258c strains and its complemented strain in 7H9 medium.

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Lin et al.

FIG 7 M. tuberculosis ⌬Rv1258c strain survival profile in resting and activated macrophages. Resting (A) and activated (50 ng/ml of TNF and 100 U/ml of IFN-␥)
(B) murine bone marrow-derived macrophages (BMMOs) were infected with M. tuberculosis strains at an MOI of 2. (C) Retinoic acid and vitamin D
(RAVD)-activated THP-1 macrophages were infected at an MOI of 5. After 45 min of incubation, the infected cells were washed with PBS to remove extracellular
bacteria (day 0 postinfection). At the indicated time points, the infected cells were lysed and viable CFU were recovered on 7H11 agar and enumerated. *, P ⬍
0.05. Data shown are means ⫾ SD from quadruplicates and are representative of two independent experiments.

hand, a large number (255) of iLiveE genes did not overlap any of as assessed by real-time PCR (data not shown). Furthermore,
the in vitro models considered in this meta-analysis and include qRT-PCR analysis showed that expression of Rv1258c was re-
genes involved in virulence detoxification pathways, cell wall pro- stored in the complemented strain Ox-Rv1258c with a 22-fold
cesses, and intermediary metabolism (see Table S4). It is plausible increase in transcriptional activity compared to the parental ex-
that these genes are modulated by lysosome-specific stimuli that pression, likely due to the use of the strong mycobacterial hsp60
are not represented in the other in vitro models. The iLivE model promoter (Fig. 6C). The in vitro fitness of both the mutant and
therefore allows investigation of M. tuberculosis responses specific complemented strains was similar to that of the parental strain
to a relevant and defined intramacrophage microenvironment, (Fig. 6D).
where the identification and biochemical characterization of host To confirm the role of Rv1258c during macrophage infection
molecules with antimycobactericidal activity can be undertaken that was previously reported (71), resting and activated (100 U/ml
(27). IFN-␥ and 50 ng/ml TNF) BMMOs were infected with WT,
Validation of the LivE model. To support the relevance of our ⌬Rv1258c, and complemented strains. A marked reduction in
transcriptomics approach to study M. tuberculosis responses to the bacterial load of approximately one log was observed with the
lysosomal environment, we selected one gene candidate, Rv1258c, ⌬Rv1258c mutant compared to the WT at 2 to 3 days postinfection
which was found highly expressed under iLivE conditions and in both resting and activated BMMOs, followed by a restoration to
functionally relevant as a membrane Tap-like efflux pump (84– parental levels at the later phase of infection (Fig. 7A and B). Pa-
86). A previous study reported that M. tuberculosis transposon rental infection profiles were observed with the complemented
mutants of Rv1258c displayed reduced viability at 96 h postmac- strain Ox-Rv1258c. The attenuation pattern observed in both rest-
rophage infection (71). However, the authors did not confirm the ing and activated macrophages suggests that Rv1258c plays a role
observed attenuated phenotype by complementation study. Here, in M. tuberculosis survival regardless of the macrophage activation
we constructed an M. tuberculosis ⌬Rv1258c mutant by homolo- status. Furthermore, the transient nature of the attenuated phe-
gous recombination and its complemented strain, Ox-Rv1258c, by notype supports that Rv1258c plays a critical role during the initial
introducing the Rv1258c ORF under the expression of the consti- phase of infection. To further demonstrate the relevance of our
tutive hsp60 promoter into the ⌬Rv1258c mutant (Fig. 6A). Dele- observations, infection profiles of the WT, ⌬Rv1258c, and Ox-
tion of Rv1258c was confirmed by Southern blotting (Fig. 6B) and Rv1258c strains were determined in human monocyte-like THP-1
did not affect the transcription of the downstream gene Rv1257c, cells that were activated by retinoic acid and vitamin D3 prior to

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 M. tuberculosis Gene Response to Lysosomal Stress

FIG 8 Susceptibility of ⌬Rv1258c and Ox-Rv1258c strains to SF and human antimicrobial peptides. (A) Mid-log-phase cultures of M. tuberculosis parental (WT),
⌬Rv1258c, and Ox-Rv1258c (complemented) strains were coincubated with 37 ␮g/ml SF for 24 h. (B) Mid-log-phase culture of the ⌬Rv1258c strain was
coincubated with 37 ␮g/ml SF for 24 h and 1 ␮g/ml CCCP. (C) WT, ⌬Rv1258c, and Ox-Rv1258c strains were coincubated with LL-37 at 30 ␮g/ml for 72 h. The
bacterial suspensions were then plated on 7H11, and CFU were determined after incubation at 37°C for 3 weeks. Data are expressed as the percentage of viable
CFU when incubated in respective assay buffers (SF buffer and 0.1% acetic acid for LL-37). Data are the means ⫾ SD from triplicates and are representative of
two independent experiments. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001; ****, P ⬍ 0.0001.

infection (34). Similar to the BMMO infection profile, an initial CCCP is expected to abrogate its activity. We observed that resis-
drop in viability was observed with the ⌬Rv1258c strain at day 1 tance to SF killing was comparable to that obtained in the absence
postinfection, but the bacilli’s replicative ability was quickly re- of CCCP (Fig. 8B). In contrast, the enhanced resistance to strep-
stored from day 2 onwards, possibly reflecting a differential envi- tomycin of Ox-Rv1258c was abrogated in the presence of CCCP,
ronmental pressure between murine and human macrophages confirming the functionality of the Tap efflux pump in this M.
(Fig. 7C). The complemented strain restored partially parental tuberculosis strain (Table 4). Together, these data suggest that the
levels of growth. mechanism(s) by which Rv1258c-encoded Tap is involved in M.
To further examine the role of Rv1258c when M. tuberculosis tuberculosis resistance to lysosomal killing does not rely on its
encounters the lysosomal microenvironment, susceptibility to efflux pump activity. Mycobacterial efflux pumps have been de-
lysosomal SF killing was compared among WT, ⌬Rv1258c mu- scribed as part of the coupled biosynthesis/export machinery for
tant, and complemented strains. Incubation with 37 ␮g/ml SF for mycobacterial cell wall components, and their respective mutants
24 h drastically reduced the viability of the ⌬Rv1258c mutant to displayed defective growth in macrophage and mouse models
3.3%, while the WT strain retained 17% viability (Fig. 8A). In (87). One could speculate that the absence of membrane-associ-
contrast, Ox-Rv1258c exhibited greater resistance to SF killing
than the WT, with 52% viability, correlating with the greater ex-
pression level of Rv1258c measured in the Ox-Rv1258c strain (Fig.
8A). Our results support a role for Rv1258c in M. tuberculosis TABLE 4 Susceptibility of M. tuberculosis WT and Ox-Rv1258c strains
to streptomycin
survival during exposure to the lysosomal content. Previous liter-
ature proposed that the Tap efflux pump activity helps mycobac- MICa (␮g/ml)
teria cope with the hostile environment by pumping out host- Treatment WT Ox-Rv1258c strain
derived antimicrobial molecules (84–86). To test whether the Tap Streptomycin 0.3 3.7
efflux pump activity is involved in the M. tuberculosis response to Streptomycin ⫹ CCCP 0.5 0.8
SF killing, the Ox-Rv1258c strain was incubated with SF in the a
The MIC of streptomycin was assayed over a range of 2-fold dilutions of the
presence of the proton motive force inhibitor CCCP. Since the compound and in the presence or absence of 1 ␮g/ml CCCP. Data shown are
Tap efflux pump requires energy to function, the presence of representative of two independent experiments.

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Lin et al.

ated Tap in M. tuberculosis results in altered cell wall composition This work was supported by the Singapore-MIT Alliance Infectious
that may render the bacterium more permeable/vulnerable to an- Disease Interdisciplinary Research Group (SMART-ID-IRG).
timicrobial compounds and compromise its cell wall-associated The funders had no role in study design, data collection and interpre-
virulence, thereby impairing survival of this pathogen within its tation, or the decision to submit the work for publication. We have no
mammalian cell host. conflicts of interest to declare.
Previous work suggested that ubiquitin-derived peptides iso-
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