Molecular Virulence Mechanisms of Mycobacterium bovis 113
variable C terminus, suggesting these genes as a
source of variation that can be acted on by selec-
tive pressures and allowing for adaptation to
changing environments or hosts (Sreevatsan
et al., 1997; Cole et al., 1998).
A wide variety of functions have been asso-
ciated with these proteins, with the majority
associated with the cell envelope (Espitia et al.,
1999; Brennan et al., 2001; Banu et al., 2002).
Rv1759c (a PE-PGRS protein), binds fibronectin
in M. tuberculosis, suggesting a role in tissue tro-
pism (Espitia et al., 1999), while the orthologue
in M. bovis is a pseudogene. PE-PGRS33 is a sur-
face protein involved in the formation of myco-
bacterial aggregates. When it was mutated in
M. bovis BCG, the resulting mutant had signifi-
cantly reduced growth (Delogu et al., 2004;
Cascioferro et al., 2007). PE-PGRS30 appears to
have a role in arresting phagosomal maturation,
as when it was knocked out in M. tuberculosis
there was a marked decrease in intracellular
bacterial replication, as well as decreased lung
pathology in mice infected with the mutant
( Iantomasi et al., 2012). Some of the PE/PPE
genes are found in clusters with ESAT-6 like
genes (Gey van Pittius et al., 2006). There are
two PE/PPE genes located in the RD1 locus,
Rv3872 gene and Rv3873, and it has been sug-
gested that these are involved in the transport of
ESAT-6 and CFP-10 out of the cell (Guinn et al.,
2004). Indeed, the linkage of ESX secretion sys-
tems with PE/PPE export has been reported by a
number of groups, for example, ESX-3 exporting
PE5, PE15 and PE20 (Tufariello et al., 2016),
while secretion of PE10 via ESX-5 is vital for
capsule integrity and virulence (Ates et al.,
2016). There are also numerous PE/PPE genes
whose expression is upregulated at different
stages during infection. During acute phases
of macrophage infection with M. tuberculosis
there is increased expression of several PE/PPE
genes (Rv0834c, Rv3097c, Rv1361c, Rv0977,
Rv1840c) (Triccas et al., 1999; Dubnau et al.,
2002; Srivastava et al., 2007). There is also evi-
dence that expression of these genes vary
between M. bovis and M. tuberculosis (Golby
et al., 2007). Further research is required in this
area to try and clarify the role these proteins play
in mycobacterial virulence, and what effect
their variation may have in mediating host
interactions.
8.6 Regulatory genesAlthough changes in individual genes may
attenuate growth or virulence in some way,
more significant changes can be identified by
looking at alterations in genes coding for regula-
tory proteins that are involved in the expression
of multiple genes. Two of these that have been
identified as particularly important for gene
expression and phenotypic differences between
M. bovis and M. tuberculosis are the PhoPR
regulatory system and the RskA-SigK regulon.8.6.1 PhoPRPhoPR is a two-component regulatory system
consisting of the PhoP response regulator and
PhoR sensor kinase components (Perez et al.,
2001; Lee et al., 2008). It has been demonstrated
that PhoPR is an important regulator of viru-
lence for M. tuberculosis, where it is involved in
the regulation of genes for sulfolipid and di- and
polyacyltrehaloses biosynthesis and secretion of
ESAT-6. Three mutations in PhoPR in M. africa-
num L6 (a human strain) and animal-adapted
species, including M. bovis, results in reduced
expression of PhoP in these species (Gonzalo-
Asensio et al., 2014). Introducing these muta-
tions into M. tuberculosis results in a recombinant
strain with reduced virulence in both human
macrophages in vitro and in infected mice. When
the mutation was corrected in M. bovis to
the M. tuberculosis allele, lipid secretion was
enhanced but there was no enhanced ESAT-6
secretion (Gonzalo-Asensio et al., 2014). It was
instead shown that the RD8 deletion in M. bovis
restored ESAT-6 secretion via a PhoPR-
independent pathway, hence suggesting a selec-
tive advantage for the loss of RD8. A strain of
M. bovis that was circulating between patients in
a Spanish HIV ward was shown to have an
IS 6110 insertion upstream of the PhoPR
locus, leading to increased PhoPR expression,
increased virulence and improved transmission
between humans, the latter being a trait that is
unusual in M. bovis infections. The loss of PhoPR
activity in M. bovis can therefore potentially
explain why this species, despite its high degree
of genetic similarity to M. tuberculosis, cannot