114 A. Smyth and S.V. Gordon
sustain an infection in human populations. Loss
of PhoPR activity altered the lipid profile of
these strains and decreased their fitness for
transmission between human hosts. Further
study into the PhoPR genotype will identify
whether mutation of this locus was beneficial for
M. bovis infection of animal hosts, and/or what
further adaptations were selected for in the after-
math to sustain M. bovis virulence in diverse ani-
mal hosts.
8.6.2 RskA-SigK regulonOne of the most consistent and significant
differences between M. bovis and M. tuberculosis
is in the levels of the secreted protein MPB70
and its membrane-bound homologue MPB83.
The genes encoding these proteins share 63%
sequence identity; however, MPB70 has no post-
translational modifications whereas MPB83 is
glycosylated and associated to the cell envelope
(Wiker et al., 1998). MPB70 first came to atten-
tion as the most dominant protein in the culture
filtrate of M. bovis and some BCG strains, but
produced at much lower levels in M. tuberculosis
(Nagai et al., 1981, 1986, 1991; Golby et al.,
2007). Later work determined that although
levels of these proteins produced by M. tuberculo-
sis are low under normal in vitro conditions,
their genes are induced during intracellular
growth (Schnappinger et al., 2003). Further
study of MPB70 identified other mycobacterial
species that had similar MPB70 levels to M. bovis
such as M. caprae (the goat bacillus) and M. ory-
gis (the oryx bacillus).
Additional research sought to explain the
molecular basis for variation in the expression of
these genes. It was first identified that the genes
MPB70 and MPB83 were under the control of
the sigma K factor (SigK). Further work recog-
nized that several other genes under this regulon
were also expressed to a much higher degree in
M. bovis than other MTBC species (Mb0455c,
Mb0456c, Mb0457c, dipZ, Mb2901, and
Mb2902c) (Golby et al., 2007). Study of SigK
indicated that the gene was identical across
the MTBC; however, when studying the
genes around SigK it was found that Rv0444c,
encoding a potential anti-sigma factor, showed
sequence variation across the complex
(Said-Salim et al., 2006). In M. bovis and M. cap-
rae, two non-synonymous SNPs were found in
Rv0444c, C320T and C551T, and as a result the
amino acids encoded changed from glycine to
aspartic acid and glutamic acid, respectively.
Additionally, a different non-synonymous SNP
was found in M. orygis, G698C, resulting in the
stop codon being replaced by a serine. Both of
these changes affected the function of the anti-
sigma factor, subsequently designated RskA,
meaning that negative control of SigK expres-
sion was lost in these strains and hence driving
constitutive expression of the SigK regulon
(Said-Salim et al., 2006).
The high level of expression of the SigK reg-
ulon would result in substantial energy costs.
Supporting this is the fact that ‘late’ strains of
BCG (e.g. BCG Pasteur), derived by repeated sub-
culture of M. bovis, lost high-level expression of
the SigK regulon via a SNP in the start codon
of SigK, and as a result show increased growth
rates compared to ‘early’ BCG strains that retain
high-level SigK expression (e.g. BCG Tokyo or
Russia) (Charlet et al., 2005). The fact that dys-
regulation of the SigK regulon occurs in some
animal-adapted MTBC species, and that it has
arisen in different species via independent muta-
tions, indicates that there is a selective advantage
for the overall success of some animal-adapted
species to overexpress the SigK regulon.
Many of the genes of the SigK regulon are
membrane associated but no particular role for
them in pathogenesis or transmission has been
identified. The focus has been primarily on
MPB70 and MPB83, although to date no strong
conclusions on their function has been made.
The structure of both has been resolved and
they have a novel fold similar to that of fascilin
domain proteins, which in other bacteria is nor-
mally implicated in protein–protein interactions
(Zinn et al., 1988; Wiker et al., 1998; Carr et al.,
2003). The studies that have been done primar-
ily focus on the role of MPB70 and MPB83 as
immunomodulatory proteins. MPB83 has been
indicated to have some immunostimulatory
properties as a TLR1/2 agonist and it has been
indicated that it can induce TNFα and MMP-9 in
human monocyte cell lines, and that blocking of
TLR1/2 receptors with antibodies caused this
response to be lost (Chambers et al., 2010).
Other experiments on murine cells also indicate
that MPB83 can induce secretion of TNFα, IL-6,