Molecular Virulence Mechanisms of Mycobacterium bovis 111
not the case and that PDIM may play a signifi-
cant role in the acute phase of the infection and
may aid in mediating interaction between the
pathogen and the host macrophage, and may in
fact encourage phagocytosis of the mycobacte-
ria (Pethe et al., 2004; Stewart et al., 2005). One
significant consideration for PDIM is that it tends
to be lost when the mycobacteria are repeatedly
sub-cultured in vitro (Domenech and Reed,
2009). Once PDIM is lost, the bacterium seems
to gain an advantage in growth speed, perhaps
due to increased membrane permeability allow-
ing more rapid diffusion of nutrients, meaning
the PDIM-negative bacteria dominate in culture
over time. This is important to consider as it has
been indicated previously that attenuation of
certain mutant knock-outs (Kos) may not be
due to inactivation of the gene in question,
but instead due to selection of mutants in PDIM-
negative backgrounds. Research into the direct
effects of PDIM seem to indicate that it has a role
in receptor-dependent phagocytosis of mycobac-
teria, and that it also may influence prevention
of phagosomal acidification (Astarie-Dequeker
et al., 2009). There are also studies showing that
it may contribute to bacterial growth and helps
resist nitric oxide dependent killing, as well as
playing a role in mediating the host immune
response, as it was shown that in a PDIM KO
there were increased levels of TNFα and IL-6
produced by murine macrophages and dendritic
cells in vitro (Rousseau et al., 2004). The major-
ity of these studies were performed using
M. tuberculosis, but there are also a number of
studies showing that loss of PDIM in M. bovis
similarly effects its virulence (Hotter et al., 2005;
Hotter and Collins, 2011).
M. bovis and M. tuberculosis both have simi-
lar levels of PDIM on their outer surface; how-
ever, certain genes that regulate synthesis and
transport of PDIM are differentially regulated
between the two (Golby et al., 2007). One study
looking at the gene expression differences
between M. bovis and M. tuberculosis when cul-
tured in vitro showed that M. bovis had much
higher expression of the lppX-pks1 genes (Golby
et al., 2007). These have been shown to be
involved in the transport and synthesis of PDIM;
however, there was no significant change in
PDIM levels, so it was suggested that this change
may reflect synthesis of PDIM-derived phenolic
glycolipids (PGL). PGL compounds are not
produced by H37Rv, the type strain of M. tuber-
culosis and the majority of clinical isolates, but
are found in M. bovis and certain Beijing-lineage
M. tuberculosis strains, with monoglycosylated
mycoside B the major PGL in M. bovis (Brennan,
2003; Malaga et al., 2008). The loss of PGLs
from many M. tuberculosis strains is caused by a
frameshift mutation in the pks1 gene, splitting it
into two genes pks1 and pks15 (Constant et al.,
2002). The M. tuberculosis strains that do have
PGLs on their surface are considered hyperviru-
lent, or more specifically hyper-lethal (Reed
et al., 2004); loss of PGL from these strains
decreases their virulence as evidenced by
increased survival of mice infected with these
variants (Reed et al., 2004).
Counter to the PGL example, sulfolipids are
trehalose-containing glycolipids that are only
expressed by M. tuberculosis and are not found
in M. bovis (Brennan, 2003). These lipids appear
to mediate pro-inflammatory responses that
aid M. tuberculosis during infection, and that
although sulfolipid KOs are not attenuated in a
murine model, there was a decrease in virulence
when human macrophages were used as
the infection readout (Gilmore et al., 2012).
Increased research into the function of sulfolip-
ids will clarify their potential to act as species-
specific virulence factors.
There are numerous cell envelope-
associated proteins whose function is still
unknown, or whose link to virulence is unclear,
but it is highly likely that the diversity of the cell
surface components will contribute to the varia-
tion in host preference, virulence and transmis-
sion of M. bovis.8.4 Secretory SystemsThe secretory systems of the mycobacteria are
highly important as a result of the near impen-
etrable cell wall barrier. Specialized systems are
required for protein and lipid secretion and the
uptake of small molecules. M. bovis, in common
with the other members of the complex, has the
common general secretion system, also known
as the Sec secretion system, consisting of a five-
part membrane complex and an ATPase that
recognizes the N-terminal signal sequence on
unfolded proteins (Braunstein et al., 2001). This