108 A. Smyth and S.V. Gordon
M. tuberculosis H37Rv and M. bovis 2122/97,
serve to set the scene. The genome sequences of
both M. bovis and M. tuberculosis are collinear
with no evidence of duplications or transloca-
tions, with the M. bovis AF2122/97 sequence
4 345 492 base pairs (bp) long and M. tuberculo-
sis H37Rv strain 4 411 532 bp (Garnier et al.,
2003). There are no unique genes present in
M. bovis that are not found in M. tuberculosis
strains; however, nine RD were deleted from
M. bovis explaining its reduced genome size.
A total of 2437 SNPs were also identified when
comparing the sequencing data of M. bovis
2122/97 and M. tuberculosis H37Rv, of which
769 were non-synonymous. Although genetic
coding potential has been lost from M. bovis as
compared to M. tuberculosis, it is still virulent
and can infect humans, although its ability to
transmit between humans is limited (Magnus,
1966). It is thought that the regions lost from
M. bovis may mostly be the removal of function-
ally redundant regions, although why with a
reduced repertoire of genes M. bovis shows a
wider range of host preferences is not under-
stood. Further understanding of the regulation
of genes and the responses these bacteria have to
changing environments could have valuable
insight into defining the ability of M. bovis to
infect and sustain in diverse hosts. The most sig-
nificant changes seem to be found in regions
coding for cell wall components and secreted
proteins, indicating that it is the interaction with
the host that is most effected by the changes in
the genome.
In addition to comparing M. bovis to
M. tuberculosis, there is an excellent source for
comparison with an avirulent M. bovis derived
strain. Bacillus Calmette–Guérin (BCG) is the
attenuated vaccine strain that has been used
worldwide as a TB preventative control tool.
Despite being the most widely used vaccine in
the world, questions about its efficacy remain.
Generated by repeated culture of M. bovis on
potato slices soaked in glycerol and ox bile, this
generated a new strain that was no longer able
to cause disease in diverse animal hosts
(Calmette, 1931). The precise molecular events
that lead to the attenuation of BCG, however,
remained elusive. A study of the genetic differ-
ences between virulent M. bovis strains and BCG
using subtractive genomic hybridization identi-
fied three large deletions between M. bovis and
BCG: RD1, RD2 and RD3 (Mahairas et al., 1996),
with RD1 the only one of these deletions missing
from all BCG strains and found in every virulent
M. bovis and M. tuberculosis strain (Mostowy
et al., 2002; Brosch et al., 2002). The role of the
genes coded for by RD1–RD3 is discussed in
more detail below.8.2.1 RD1–RD3The loss of the RD1 locus from BCG was identi-
fied as playing a key role in the loss of virulence
of the vaccine strain. Complementation of BCG
with RD1 restores virulence to the vaccine strain
(Pym et al., 2003; Majlessi et al., 2005), although
not to the full degree seen in M. bovis or M. tuber-
culosis. When RD1 is deleted from M. tuberculosis
the strain is also attenuated, confirming the key
role of the genes encoded by this region in viru-
lence (Lewis et al., 2003). RD1 encodes a type
VII secretion system (T7SS) that secretes a range
of proteins including the potent T-cell antigens
ESAT-6 and CFP-10 (Berthet et al., 1998b; Bitter
et al., 2009) and hence is also known as the
ESAT6 secretion system 1 (ESX-1). A site distal
from RD1, the espACD locus is also important for
the activity of the ESX-1 system, although this is
still present in BCG strains.
ESAT-6 and CFP-10 are small secreted pro-
teins (6 kDa and 10 kDa, respectively) and both
belong to the ESAT-6 family of proteins, a family
of secreted proteins with 23 members in the
mycobacterial genome (Brodin et al., 2004).
These genes occur in tandem pairs throughout
the genome and although little is known about
their function it is theorized that once expressed
they interact to form protein complexes. Inter-
estingly, six of the ESAT-6 protein family
are missing or altered in M. bovis (Rv2346c,
Rv2347c, Rv3619c, Rv3620c, Rv3890c and
Rv3905c) (Garnier et al., 2003). The effects of
these losses have not been studied, and what role
they may play in mycobacterial virulence is not
understood as of yet. The precise role of ESAT-6
and CFP-10 in the establishment of infection is
still not completely understood, but they have a
key role to play in the virulence of mycobacteria.
Both proteins have been shown to have immu-
nogenic properties and can induce T-cell
responses across multiple species (Aagaard et al.,