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Evolution of virulence in the Mycobacterium
tuberculosis complex
Mickael Orgeur and Roland Brosch
Mycobacterium tuberculosis, the causative agent of human
tuberculosis is one of the most widely spread human
pathogens. It has succeeded to infect a quarter of the global
human population by developing most sophisticated ways to
circumvent innate and adaptive immune defences. This highly
specialized, major human pathogen has evolved from a pool of
ancestral environmental mycobacteria, whose extant
representatives are known under the name of Mycobacterium
canettii. Recent whole genome analyses in combination with
different phenotypic screens have provided key insights into
the evolution of M. tuberculosis and closely related members
regrouped in the M. tuberculosis complex (MTBC). They
have also elucidated novel virulence determinants that are
essential for these obligate pathogens. In this review, we
present the most recent evolutionary models of the MTBC and
various factors that have contributed to the outstanding
evolutionary success of the tuberculosis agent.
Address
Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics,
75015 Paris, France
Corresponding author: Brosch, Roland (roland.brosch@pasteur.fr)
Current Opinion in Microbiology 2018, 41:68–75
This review comes from a themed issue on HPI: Evolution & regula-
tion of HPI
Edited by Joan Mecsas and Carmen Buchrieser
https://doi.org/10.1016/j.mib.2017.11.021
1369-5274/ã Elsevier Ltd. All rights reserved.
Introduction
The history of tuberculosis is full of countless human
tragedies, and even today tuberculosis represents the
infectious disease that claims the most victims worldwide.
Tuberculosis, previously also known as phthisis, con-
sumption, or white plague, was for long considered as
an inherited condition until the ground-breaking discov-
ery of Robert Koch in 1882, who defined it as one of the
first infectious diseases that was caused by a bacterial
pathogen, named Mycobacterium tuberculosis. During more
than 135 years, dedicated studies have considerably
enriched our knowledge on many aspects of the pathogen
and its interaction with the host. In recent years, the
Current Opinion in Microbiology 2018, 41:68–75
dramatic increase in the generation of genomic data has
allowed unprecedented insights to be gained into the
evolution of tuberculosis-causing mycobacteria and the
mechanisms employed by M. tuberculosis to circumvent its
elimination by the immune system during infection. A
wide range of potential mycobacterial virulence factors
has been highlighted by genome-wide transposon inser-
tion-based screening approaches and/or dedicated gene
knock-out/knock-in strategies in various infection mod-
els. For many of them, the available information on the
biological mechanisms and function in the infection
process is still scarce. However, for some others, such
as the ESX-1 secretion system, deeper insights have been
gained, which are discussed here together with associated
aspects that seem to impact the evolution and distribution
of mycobacterial strain populations.
Evolution of the clonal M. tuberculosis
complex from a recombinogenic
Mycobacterium canettii-like strain pool
M. tuberculosis is a bacterium with a parasitic lifecycle that
needs to cause pulmonary lesions in patients in order to
get efficiently spread to new individuals through cough-
ing up bacteria-loaden droplets and aerosols. According to
most recent estimations, a quarter of the global human
population is infected with this pathogen [1]. New
insights into the factors that have allowed M. tuberculosis
to become such a powerful and efficaciously spread
pathogen are thus of key interest. Comparison of M.
tuberculosis with closely related mycobacteria that show
differences in the mode of transmission, the host spec-
trum and/or the geographical distribution, have the
potential to provide essential information on the evolu-
tionary trajectory of M. tuberculosis towards increasing
pathogenicity and spread.
Recent genome-based studies of various tuberculosis-
causing mycobacteria have found evidence that the M.
tuberculosis complex (MTBC) likely emerged as a single
clonal group from a pool of recombinogenic mycobacterial
strains that resembled extant M. canettii strains [2,3].
These latter strains are named after Georges Canetti,
who was a renowned tuberculosis specialist at the Institut
Pasteur in Paris in the 1960s and first isolated a strain
showing unusual, smooth colony morphology from a
tuberculosis patient [4]. M. canettii strains, which are also
known as smooth tubercle bacilli (STB), are very rare and
were primarily isolated from patients at the Bouffard
French Military Hospital and the Paul Faure Anti-Tuber-
culosis Centre in Djibouti [5,6], or from patients with
www.sciencedirect.com

other connections to the geographical region around the
Horn of Africa [4]. Although the less than 100 M. canettii
strains that are known today were all isolated from human
infections, M. canettii strains are considered as potential
environmental mycobacteria because human-to-human
transmission or an epidemiological link between outbreak
strains could not be demonstrated so far [5,6]. Thus,
contamination and infection of individuals likely have
occurred through contact with a yet unknown source. The
finding that M. canettii strains show highly recombino-
genic genomes [2,7] points to a putative environmental
source where frequent contact of different strains might
allow inter-strain DNA transfer to be efficiently estab-
lished. This hypothesis based on in silico data was recently
strengthened by the experimental demonstration of hori-
zontal transfer of multiple, large chromosomal DNA
fragments from one M. canettii strain (strain A, donor)
to another M. canettii strain (strain L, recipient) under
laboratory conditions [8
]. The same study also found that
such DNA transfer did not occur in M. tuberculosis strains
despite extensive attempts. This suggests that horizontal
gene transfer has played an important role in the early
stages of the evolution of tuberculosis-causing mycobac-
teria, while the faculty for inter-strain transfer of chromo-
somal DNA seems to have been lost later in the clonal
MTBC [8
]. Another detail arguing that the clonal mem-
bers of the MTBC have originally evolved as a single
lineage from an M. canettii-like progenitor pool is the
finding that M. canettii strains produce lipooligosacchar-
ides (LOS) [4,9
], similar to environmental mycobacterial
species such as Mycobacterium marinum [10] or Mycobacte-
rium kansasii [11]. In contrast, the clonal members of the
MTBC have lost the ability of LOS synthesis despite the
presence of a large portion of the LOS synthesis gene
locus remaining in the genome of these strains. A recent
study has shown that homologous recombination between
two adjacent pks5 genes leads to abrogation of LOS
production and appearance of rough colony variants of
M. canettii, which depict a genetic organisation in this
locus similar to that of M. tuberculosis [9
]. Interestingly,
the rough M. canettii variants showed increased virulence
in the guinea pig model of infection, and increased levels
of TLR2-mediated signalling in THP-1 cells [9
] This
suggests that loss of LOS production during the evolution
of tuberculosis-causing mycobacteria likely has contrib-
uted to the emergence of a more potent and more virulent
lineage, which then underwent clonal expansion and only
minor differentiation. This clonal group is now repre-
sented by 7 lineages of closely related M. tuberculosis (L1–
L4, L7) and Mycobacterium africanum (L5–L6) strains that
cause tuberculosis in humans, as well as animal-adapted
strains that branch next to L6 M. africanum and affect
various mammalian animal species (Figure 1). Several
recent studies have postulated that M. tuberculosis has
undergone its early evolution on the African continent.
This hypothesis is based on the finding that, in addition to
M. canettii, representative strains from all 7 extant lineages
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Mycobacterial virulence evolution Orgeur and Brosch 69
of human isolates, as well as most members of the animal
lineages, can be found in various regions of Africa [12
,13–
16]. The most ancestral lineages of human MTBC mem-
bers are represented by the M. africanum lineages L5 and
L6 and the M. tuberculosis lineages L1 and L7 that have
diverged before the deletion of the M. tuberculosis specific
genomic region TbD1 occurred (Figure 1). The deletion
of the TbD1 region represents a characteristic molecular
event and evolutionary bottleneck that defines the origin
of the ‘modern’ L2, L3 and L4 M. tuberculosis lineages,
which are considered as the most recently evolved M.
tuberculosis strains [12
,13,14,17]. Interestingly, the latter
lineages represent the geographically most widespread
strain families, including the Euro-American (L4) lineage
[18] and the so-called Beijing strains (L2) [19
,20
], which
have spread to many countries of the world. The question
why some stain lineages remained geographically
restricted, whereas others have developed into globally
distributed epidemic clones, remains a subject of debate
and lacks for the moment a clear answer, although most
recent findings may provide partial explanations [18,21
].
However, to enrich this discussion, it might also be worth
taking results from molecular investigations on the line-
age of animal-adapted strains into consideration.
Evolutionary particularities in the animal-
adapted members of the MTBC
Genetic analyses have shown that the animal-adapted
strains share a last common ancestor with the L6 M.
africanum strains that belongs to the RD9-deleted branch
of the MTBC, which additionally has deleted regions
RD7, RD8 and RD10 [15,17,22

,23
] (Figure 1). The
RD9-deleted lineage of tubercle bacilli has also specifi-
cally accumulated mutations in the genetic region encod-
ing the PhoP/PhoR two component system, whose regu-
latory network of more than 100 genes also includes many
genes involved in virulence [22

,24]. These mutations
include a GGT ! ATT codon change in the PhoR sensor
that occurred before the branching of the L5 M. africanum
lineage and is thus present in all M. africanum and animal-
adapted strains (Figure 1). In addition, a mutation in the
phoP promoter region (nt 48 G ! A) is present in L6 M.
africanum and animal-adapted strains (Figure 1). It was
demonstrated by phoP/R allele switching experiments
between M. tuberculosis and Mycobacterium bovis, that these
mutations in the phoP/R region are responsible for a much
lower production of the biologically active lipids of the
polyacyltrehalose (PAT) and sulfolipid (SL) families
[22

]. Moreover, a strong decrease in the presence of
the ESX-1 type VII secretion substrates EsxA (also
known as ESAT-6) and EsxB (also known as CFP-10)
was observed in the culture supernatants when the
mutated allele was transferred into an M. tuberculosis
genetic background. This latter effect is linked to the
impact of PhoP/R-mediated regulation of EspR [25,26],
Lsr2 [27] and the two component system MprA/B [28] on
the expression of the ESX-1 secretion-associated proteins
Current Opinion in Microbiology 2018, 41:68–75
70 HPI: Evolution & regulation of HPI

Figure 1

M. canettii (outgroup)

L1 M. tuberculosis

M. africanum L5

RD7, RD8, RD10

M. africanum L6

L7 M. tuberculosis Chimpanzee bacillus

RD9 M. microti

RD1 mic

M. pinnipedii
ancient tubercle bacillus
TbD1 phoP (from Peruvian mummier)
codon 71
GGT ATT RD12. RD13
M. orygis
phoP promoter
nt -48 G A RD4

L4 M. caprae
M. tuberculosis
L3 M. bovis
M. tuberculosis

L2 M. tuberculosis
Current Opinion in Microbiology

Phylogenetic relationships of the different MTBC lineages, based on SNPs of 261 mycobacterial genomes, adapted from reference [23
], including
additional information on deletion points of selected regions of difference (RD) [17] and selected mutations in the PhoP/PhoR mycobacterial two
component system [22

]. Note that in previous spoligotyping cluster naming schemes, L1 strains correspond to East-African-Indian (EAI) strains,
L2 strains to Beijing strains, L3 strains to Dehli/Central Asian strains (Dehli/CAS), L4 strains to Euro-American strains (T, Haarlem, LAM, S, X), L5
to M. africanum 1 and L6 to M. africanum 2 strains. L7 strains are restricted to Ethiopia and the Horn of Africa region.

EspA and EspC, known to be secreted in a co-dependent
fashion with EsxA and EsxB [29,30,31
]. However, this
regulation cascade of EsxA and EsxB secretion via co-
dependence with EspA and EspC can also be short cut,
which is the case for M. africanum L6 strains and all
animal-adapted strains [22

]. In these strains, the RD8
genomic region just upstream of the espACD operon,
which contains various binding sites for transcriptional
regulators, is deleted (DRD8) (Figure 2). This DRD8-
caused genomic conformation thus allows EsxA/B secre-
tion to be achieved despite strongly reduced PhoP/R
activity [22

]. The deletion of the RD8 region and the
corresponding gain of EsxA/B secretion, independent of
the PhoP/R, Lsr2 and MprA/B regulatory systems, thus
represent key events in the evolution of strains belonging
to the lineage of RD9-deleted tubercle bacilli, that is, M.
africanum and animal-adapted strains. It appears that this
regained robustness in EsxA/B secretion might have
created the conditions for successful infection of new
mammalian hosts (animal-adapted strains), while retain-
ing the faculty to successfully infect humans (L6
M. africanum strains). In this respect, it is remarkable
that the human-adapted M. tuberculosis strains have been
shown to be attenuated in cattle [32], an observation that
was already made more than hundred years ago by Emile
von Behring, Robert Koch and Theobald Smith [32]. On
the other hand, it was also shown that M. bovis strains and
other animal-adapted lineages can infect humans and
even cause disease. However, from epidemiological data,
it is clear that most of these infections represent spill-over
events that do not have the potential to be carried on by
aerosol transmission to other humans and cause epi-
demics [22

,33]. Discussions on the plausible reasons
for the host preferences within the MTBC remain thus
animated [34,35], highlighting the need of additional
molecular work that can clarify the bacterial and host
determinants involved.
The ESX-1 system as one of the key virulence
elements of tubercle bacilli
The ESX-1 system represents one such bacterial key
element involved in the interaction between pathogenic

Current Opinion in Microbiology 2018, 41:68–75 www.sciencedirect.com


Figure 2
espD espC espA IpqG
espD espC espA
4054000 4056000
espD espC espA IpqG
Deletion of the RD8 region results in shortcutting of the PhoP/R, Lsr2, and MprA/B-mediated expression control of EspA and EspC, which show
interdependence in their secretion with EsxA (ESAT-6) and EsxB (CFP-10). Schematic representation of the genetic structure at the espACD locus
in M. tuberculosis, M. africanum L5/L6 and animal-adapted tubercle bacilli (M. bovis). Red lines indicate individual SNPs identified between
pairwise-compared genomes. Blue lines indicate the boundaries of the RD8 deletion.
Image adapted from Ref. [22

].
mycobacteria and the host. Indeed, ESX-1 is needed for
phagosomal rupture and contact of the bacterium with the
host cytosol during infection [36]. It was for long consid-
ered that M. tuberculosis was an exclusive intra-vacuolar
pathogen that had the ability to inhibit the phagosomal
maturation process, thereby favouring bacterial multipli-
cation [37,38]. However, recent studies have clearly
shown that ESX-1-proficient tubercle bacilli also gained
access to the host cytosol [39,40,41
]. In contrast, the
attenuated Bacillus Calmette-Guérin (BCG) vaccine,
which has lost part of the genes encoding for the ESX-
1 secretion system due to deletion of the RD1 region,
cannot access to the host cytosol [39,40] and does not
induce cytosolic pattern recognition responses [42–44].
This difference between pathogenic M. tuberculosis and
attenuated BCG has numerous consequences for host
pathogen interaction and vaccine efficacy [43,45]. It is
likely that the RD1 deletion in BCG and the resulting loss
of ESX-1 functions has occurred during the long-term in
vitro passaging that Albert Calmette and Camille Guérin
have imposed on an originally fully virulent M. bovis
isolate at the beginning of the 20th century. Plausibly,
ESX-1 functions were not anymore required by the strain
under the in vitro conditions used, that is, growth on
medium prepared from cooked potatoes and sterile 5%
glycerinated ox-bile. However, it is intriguing that some
other members of the MTBC that cause infections in
selected mammalian animal hosts have natural deletions
in the RD1 genomic region encoding the ESX-1 system.
The best-known examples are Mycobacterium microti
strains, which were originally found as the causative agent
of severe skin lesions in wild voles, and more recently also
identified in cats and few other animal species [46]. These
strains, which form a sublineage in the animal-adapted
lineage of the MTBC, all show a deletion of 13 kb
region, overlapping the RD1 region of BCG [46]. Like
BCG, some M. microti strains were used for anti-tubercu-
losis vaccination in humans, showing an attenuated
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Mycobacterial virulence evolution Orgeur and Brosch 71
M. bovis
M. tuberculosis &
RD8 M. africanum L5
ephA Rv3618 esxV esxW PPE65 PE32 IpqG
4058000 4060000 4062000
M. africanum L6
Current Opinion in Microbiology
phenotype. Apart from M. microti, three other members
of the MTBC were reported as showing deletions in the
RD1 region, that is, Mycobacterium mungii, Mycobacterium
suricattii and the so-called Dassie bacillus [16,47,48].
These strains, which branch next to the L6 strains of
M. africanum can cause severe infections in mongooses,
mercats and dassies in Africa. For example, M. mungii
strains are transmitted by specific social communication
behaviour of mongooses, as numerous diseased animals
with mycobacterial lesions at the anal glands and nasal
planum were observed [16]. It is possible that superficial
skin infection and a contact-dependent contamination
route might provide an easier way of transmission for
ESX-1-deficient tubercle bacilli that do not have the
fitness to cause full-blown pulmonary disease. These
examples also clearly show the importance of the host
characteristics and the specific adaptations of certain
tubercle bacilli to the immune system of particular hosts.
Additional virulence factors cooperating with
ESX-1
Finally, most recent data show that the ESX-1 secretion
system acts in concert with phthiocerol dimycocerosates
(abbreviated as DIM or PDIM) to enable phagosomal
rupture during infection of host macrophages [49
]. DIM/
PDIM are known as major virulence factors of M. tuber-
culosis. It became now evident that these extractible
lipids, located in the outer membrane of slow-growing
mycobacteria, are needed for the induction of phagosomal
rupture by M. tuberculosis [49
,50]. These findings were
further confirmed by a study that used transposon inser-
tion mutants and high-throughput phenotypic multipara-
metric analyses [51]. Comparable profiles were detected
both in mutants affected in the ESX-1 system, and in
mutants affected in the synthesis or transport of DIM/
PDIM [51]. The ability to induce phagosomal rupture
thus depends on protein and lipid virulence factors that
need to act in concert. This suggests that cooperation of
Current Opinion in Microbiology 2018, 41:68–75
72 HPI: Evolution & regulation of HPI

Figure 3

M. canettii & M. tuberculosis complex

(rv C )

c)
p c

es 15c
)

rv 7)
es 14

16
10

18
11

1
(rv D

hA
(rv A
H

36

36

36
36

36

36
36

p
fts

ep
es
(rv

(rv
rv
M. kansasii

54
51

pD
pC

pA

36
36

es
es

es

rv
rv
IpqD
M. lepromatosis &

(rv3390) amiB2
M. haemophilum,

(rv1263)

M. marinum &
espD
espC espD
approximate

M. liflandii
M. leprae

espA M. tuberculosis H37Rv espC

genome coordinates espA

cmaA1
(rv3392c)
iunH
(rv3393c)
pknH
(rv1266c)
embR
(rv1267c)
Current Opinion in Microbiology

Schematic representation of the genomic integration sites of the espACD cluster in the different slow-growing mycobacterial species, known to
harbour orthologous genes of espA, espC and espD. Note that the orthologous flanking genes of the espACD cluster identified in the various
species, depicted in black colour, refer to the M. tuberculosis H37Rv gene nomenclature and genomic localization. These genes may be localized
in different genomic positions in the various mycobacterial species concerned.
Image taken from Ref. [52].

these — in principle independent features — got estab-
lished during the evolution of pathogenic mycobacteria.
Interestingly, DIM/PDIM are only present in certain
slow-growing mycobacteria [49
], and the same is true
for the ESX-1-associated proteins of the EspACD cluster
(Figure 3) [52]. In addition, the induction of phagosomal
rupture by M. tuberculosis also depends on the initial
control of phagosomal acidification [41
], one of the
key mechanisms used by the host macrophages to limit
bacterial growth. We know from previous work that
M. tuberculosis can target the phagosome-associated
V-ATPase by interaction with the mycobacterial phos-
phatase PtpA [53]. However, an alternative cellular mech-
anism was recently described. Infection of macrophages
with M. tuberculosis interferes with host suppressor of
cytokine signalling (SOCS) protein functions, which leads
to ubiquitination and degradation of H+-VATPase and
shut-down of the proton pump [54
].
Concluding remarks
In conclusion, recent research has provided new insights
into the evolution of mycobacterial virulence determi-
nants, from which we can deduce that the presence of
selected virulence lipids (DIM/PDIM) in combination
with selected virulence protein secretion factors (ESX-1
system and EspACD locus) was necessary for the initial
pathogenicity of tubercle bacilli. Moreover, during the
early evolution of tubercle bacilli from a likely environ-
mental, M. canettii-like ancestor into the clonal MTBC,
the loss of LOS and the associated changes in bacterial
surface structure and immune signalling also seem to
have had an important impact on the evolutionary success
as a pathogen. These events were then followed by
lineage-specific accumulation of intra-genome insertion/
deletion events and single-nucleotide polymorphisms
(SNPs), which apparently have again changed the viru-
lence potential of selected lineages. One of the key events
for the M. africanum L6 and animal-adapted lineages was
the loss of the RD8 region, which seemingly made these
strains independent from PhoP/R regulation of ESX-1
secretion processes. Overall, it becomes clear that the
virulence phenotypes of tubercle bacilli are strongly influ-
enced by the genomic organisation of the bacterium, but
also on the host susceptibilities, which may differ from
host to host and individual to individual [55]. The tech-
nological revolution of DNA and RNA-based technologies

Current Opinion in Microbiology 2018, 41:68–75 www.sciencedirect.com


allows now to screen the transcriptional and epigenomic
profiles of the pathogen and/or the host [56
,57

]. This
will be of particular interest for future comparative studies
implicating tubercle bacilli from distant positions of the
phylogenetic tree and host cells from various mammalian
species, which shall further elucidate the specific condi-
tions where and when virulence factors and host responses
are induced.
Conflict of interest statement
None declared.
Acknowledgements
We are grateful to our colleagues who contributed to the studies that are
reviewed in this article. Support by grants from the Agence National de
Recherche (ANR-10-LABX-62-IBEID, ANR-16-CE15-0003 and ANR-16-
CE35-0009) and from the Fondation pour la Recherche Médicale
(SPF20160936136 and DEQ20130326471) are gratefully acknowledged.
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