112 A. Smyth and S.V. Gordon
system transports proteins across the inner
membrane into the periplasmic space. It has not
yet been explained how the proteins then travel
across the outer portion of the cell wall, and it is
expected that there are additional pathways
used to direct proteins across this space that are
as yet unidentified. One protein known to be
secreted through this pathway is MPB70, dis-
cussed below, a protein produced at significant
levels by M. bovis. Mycobacteria also encode a
second homologous SecA pathway termed
SecA2 (Braunstein et al., 2001; Swanson et al.,
2015). The exact role of this pathway in patho-
genesis is unknown, but its removal has negative
effects on M. tuberculosis growth in vivo (Braun-
stein et al., 2003); further work on the substrates
secreted by this pathway is needed to elucidate
the role of the secreted effectors in mycobacte-
rial virulence. Mycobacteria also possess a twin
arginine transporter (Tat) pathway that is also
found in a number of other pathogenic bacteria
where it is essential for virulence (Lee et al.,
2006). The Tat pathway translocates folded pro-
teins across the inner membrane, and its loss
compromises the ability of M. tuberculosis to
grow in vitro (Saint-Joanis et al., 2006). There is
also evidence that certain substrates secreted via
Tat, such as phosopholipase C enzymes, are
essential for full virulence of M. tuberculosis in
vivo (Raynaud et al., 2002a; McDonough et al.,
2005).
As stated above in the description of RD1,
the MTBC have a family of type VII secretion
systems. Pathogenic mycobacteria including
M. bovis possess five T7SS (Houben et al., 2014),
the best characterized of which is the previously
mentioned ESX-1, which is involved directly in
host–pathogen interactions, and whose loss is
one of the key factors of BCG attenuation
(Houben et al., 2012). The other four members
are not as well studied; ESX-2 and ESX-4 have no
known function, while ESX-3 is involved in the
balance of zinc and iron in the bacterium and in
secretion of PE and PPE proteins, an important
mycobacterial protein family discussed in more
detail below (Serafini et al., 2009; Tufariello
et al., 2016). ESX-5 has only been found in slow-
growing mycobacteria, its appearance in the
genetic record also seems to coincide with the
differentiation between slow- and fast-growing
mycobacteria (Gey Van Pittius et al., 2001). It
has also been shown that ESX-5 is essential for
the secretion of PPE and PE-PGRS (polymorphic
GC-rich repetitive sequences) proteins, a topic
we will move to next (Abdallah et al., 2009).8.5 PE/PPECertain protein families are of particular interest
due to their unique properties and their varia-
tion across the MTBC. While the members of the
MTBC show a high degree of sequence similar-
ity, greater than 99.9% in most cases, there are
two gene families present in the MTBC that are a
major source of sequence polymorphism. These
are the PE and PPE gene families, PE named for
the Pro-Glu residues found at residues 8 and 9
on the N terminus of the proteins and PPE for
the Pro-Pro-Glu motif in the same region (Cole
and Barrell, 1998; Cole, 1999). The PE family
has around 100 members in M. tuberculosis and
PPE has around 68, although the numbers vary
across the MTBC, with 10% of the coding capac-
ity of the bacteria being taken up by these gene
families. These proteins appear unique to myco-
bacteria and are found in greater abundance in
pathogenic mycobacteria. The largest family of
PE proteins is the PE-PGRS protein family, with
these proteins in particular seeming to have
some immunologically important role (Delogu
and Brennan, 2001; Sampson, 2011). The
genes for PE and PPE proteins are clustered
together throughout the genome, and it has
been shown that some are co-transcribed and
can form a stable complex. There are 40 PE/PPE
gene pairs in the M. tuberculosis genome, and 22
of these contain PE/PPE genes exclusively, sug-
gesting that these linked genes may have related
roles (Overbeek et al., 1999; Tundup et al.,
2006).
Predicted protein function based on amino
acid sequence indicated that 40 of the PE/PPE
genes have beta barrel-like amino acid sequences
(Pajon et al., 2006). Genetic analysis of M. bovis
and M. tuberculosis has shown there are blocks
of sequence variation in these regions, which
affects 29 different PE-PGRS and 28 PPE pro-
teins, resulting mostly from in-frame deletions
and insertions (Garnier et al., 2003). These pro-
teins have a conserved N terminus and a highly