Molecular Virulence Mechanisms of Mycobacterium bovis 109
2010; Arlehamn et al., 2012; Kassa et al., 2012).
The genes for ESAT-6 and CFP-10 are co-
transcribed and the proteins form a tightly bind-
ing 1:1 complex (Renshaw et al., 2002), with
transport of the ESAT-6:CFP-10 heterodimer
across the membrane dependent on the
C- terminal region of CFP-10 (Dillon et al., 2000;
Champion et al., 2006). The activity of the single
proteins has also been explored and it was shown
that while ESAT-6 destabilizes and lyses lipo-
somes, CFP-10 does not appear to have this
effect (Guinn et al., 2004). The ability of ESAT-6
to lyse the membrane appears key to the process
whereby M. tuberculosis can escape from the
phagosome into the cytosol of infected cells, as
RD1 or ESAT-6 mutants of M. tuberculosis are
defective in phagosome escape (van der Wel
et al., 2007) and the mutants do not spread to
surrounding cells (Guinn et al., 2004).
The other two deletions, RD2 and RD3, are
of lesser importance to the attenuation of BCG
and hence mycobacterial virulence. RD2 was
lost sometime during the subculture of BCG sub-
sequent to 1927, and while its loss does not fully
attenuate the bacteria it does seem to have some
contribution to virulence. RD2 contains the
mpt64 gene, which codes for a known immuno-
genic protein. Deletion of RD2 in M. tuberculosis
does not affect the growth of the bacteria, but it
does decrease the bacterial burden and histopa-
thology seen in mice (Kozak et al., 2011). Com-
plementation of the mutant with a section of
RD2 containing the mpt64 gene restored the
mutant’s phenotype to that of the wild type,
indicating some importance for this protein in
bacterial virulence (Mustafa et al., 2007). RD3 is
found in M. tuberculosis H37Rv and M. bovis but
is absent from BCG and 84% of clinical M. tuber-
culosis isolates (Mahairas et al., 1996). RD3 is a
prophage so its loss from BCG may be due to
phage excision, while its absence from many
virulent strains would indicate it is not required
for virulence.
8.3 Cell EnvelopeThe proteins, lipids and carbohydrates that lie at
the surface of the bacterial cell are clearly cru-
cial in host–pathogen interaction as they directly
interact with host cells. Changes in the lipid
profile on the cell surface therefore have signifi-
cant effects on host–pathogen interactions.
There are distinct differences in the lipids found
on the surface of M. bovis when compared to
other MTBC species, differences that could play a
role in dictating host preference. Indeed, the
only locus that is present in M. bovis and absent
from ‘modern’ M. tuberculosis lineage strains is
the TbD1 locus (Garnier et al., 2003). This con-
tains the mmpS6 gene and the 5′ region of
mmpL6. It is thought that loss of these genes
may prevent trafficking of particular lipids to the
surface of the mycobacteria, but the presence of
the locus in ‘ancient’ M. tuberculosis strains does
not mark TbD1 as a strong candidate for a role in
host preference.
One of the key features of the mycobacteria
is their distinctive waxy cell wall, which is much
thicker than that of other bacteria (Brennan,
2003). The thick waxy coat of the mycobacteria
is highly impermeable and tough, acting as a
shield against host defences but also harbouring
an array of immune modulatory compounds.
The cell wall contains covalently linked peptido-
glycan, arabinogalactan and mycolic acids gen-
erating a so-called ‘mycomembrane’. Interlinked
with the mycolic acids are a plethora of free
lipids such as phthiocerol dimycocerosate, cord
factor, sulfolipid, phenolic glycolipids and phos-
phatidylinositol mannosides. An outer capsule
of α-glucan provides the final layer. This thick
protective coating makes transport of com-
pounds into and out of the cell a complex pro-
cess involving multiple import and export
systems that have also been implicated in viru-
lence due to the transport of effectors or immu-
nomodulatory compounds. Although knocking
out the genes for certain transport components
may reduce the virulence of the organism it is
often not clear if that component directly inter-
acts with the host and invokes specific negative
effects, or if by altering single components,
larger disruptions are carried across the funda-
mental structure of the entire membrane, alter-
ing protein secretion and surface structures that
may be directly affecting the host.
A large family of secreted and surface-
exposed proteins is the Mce family, originally
termed mammalian cell entry proteins as they
were thought to be involved in cell invasion
(Arruda et al., 1993). The genes encoding the
Mce proteins are arranged into operons,