Chapter 16

Mycobacterium tuberculosis Hsp60 as a Key

Virulence Factor in Tuberculosis

Richard W. Stokes

Abstract Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a

major pathogen of man with about one-third of the world’s population being in-
fected. M. tuberculosis resides within macrophages which are members of the host’s
cell-mediated immune response that supposedly protect against bacterial invasion.
Obviously M. tuberculosis has strategies that enable it to survive in this hostile en-
vironment. Amongst the many virulence factors that M. tuberculosis possesses are
two paralogues of the ubiquitous stress protein, chaperonin (Cpn) or Hsp60 that have
been named Cpn60.1 and Cpn60.2. While cpn60.2 is an essential gene involved in
the maintenance of cell viability through “normal” chaperoning activities, cpn60.1
is non-essential and appears to have little or no involvement in protein folding ac-
tivities. Both Cpn60.1 and Cpn60.2 have varied “moonlighting” functions and can
act as secreted signaling molecules, modulators of host immunity, surface located
bacterial ligands and bacterial cell wall components. How these proteins leave the
cytosol to function extracellularly or at the surface of the bacterial cell wall is still
not clear. That they can act as bacterial virulence factors is becoming clear although
recognition of Cpn60.2 by the host may instead mediate a host defence mechanism.

16.1 Introduction

All the other chapters in this book have concentrated on the cell stress proteins of
mammals, largely Homo sapiens. However, bacteria express many homologs of the
eukaryotic cell stress proteins and there is emerging evidence that these proteins can
play a role in bacterial virulence. The vast majority of the genus, Mycobacterium,
are saprophytic species that, like other Actinomycetes, are found in soil and water.
Some species, however, are the causative agents of disease in man. While some
of these (e.g. the M. avium-intracellulare complex, M. kansasii and M. fortuitum)
are opportunistic pathogens of man, that predominantly cause disease in immuno-
compromised individuals, some species are major pathogens of man. These include
Mycobacterium tuberculosis, M. leprae and M. ulcerans of which the best known

R. W. Stokes ()
Department of Microbiology & Immunology, University of British Columbia,
Life Sciences Centre, 2504–2350 Health Sciences Mall, Vancouver,
British Columbia, V6T 1Z3, Canada
e-mail: rstokes@mail.ubc.ca

B. Henderson, A. G. Pockley (eds.), Cellular Trafficking of Cell Stress Proteins in Health 243
and Disease, Heat Shock Proteins 6, DOI 10.1007/978-94-007-4740-1_16,
© Springer Science+Business Media Dordrecht 2012
244 R. W. Stokes

and most important is M. tuberculosis (M. tb), the causative agent of tuberculosis
(TB). TB causes more death in adults than does any other single bacterial species
and it is estimated that in the year 2010 there was approximately ten million new
cases of TB (Dye and Williams 2010) and that TB is the cause of around two million
deaths worldwide (Anonymous 2009). The relatively recent emergence of multidrug
resistant and extensively drug resistant TB has become a serious problem and threat-
ens to make the disease incurable (Jain and Mondal 2008; Mitnick et al. 2008).
Unfortunately, the recent AIDS pandemic has compounded the infection rates and
morbidity of TB, due to the diminished CD4 T-cell mediated immunity of the AIDS
patient (Getahun et al. 2010). Despite the recent successes in the global treatment of
TB, it remains a devastating disease of mankind that continues to infect increasingly
higher numbers of people (Dye and Williams 2010).
Mycobacterium tuberculosis is an intracellular pathogen that generally resides
within macrophages (Ms) and is predominantly found in the lung. Ms are phago-
cytic cells that act as part of the effector arm of the host’s cell-mediated immunity.
They can kill invading microbes (though not as efficiently as do neutrophils) us-
ing a variety of mechanisms such as reactive oxygen intermediates (ROI), cationic
peptides, LRG-47 and by undergoing apoptosis. Obviously, in some cases, M. tb
survives the initial move to the lung and a subsequent intracellular environment.
There it begins to replicate which results in bacterial antigens being processed by the
host leading to the development of adaptive immunity resulting in the production of
interferon-γ (IFNγ ) by T-cells (Orme 2004; Cooper 2009; Torrado et al. 2011). The
IFNγ acts on the infected Ms to increase their ability to control bacterial replication
through what is called classical activation. M activation leads to an enhancement
of the killing mechanisms seen in resting Ms (such as ROI and apoptosis) and to
the induction of new killing mechanisms mediated by reactive nitrogen intermedi-
ates (RNI) or phagosomal maturation to lysosomes. This exposure to IFNγ activated
Ms following the development of adaptive immunity marks a change in the patho-
genesis of TB. The bacterial growth slows down drastically to the point where there
is little bacterial replication and what little there is is countered by host immunity
(Gill et al. 2009; Ehlers 2009). This coincides with a change to the physiology of
M. tb towards a so called dormant (also called latent or chronic) phase. In this state,
M. tb metabolism switches to the use of lipids instead of carbohydrates as a carbon
source and to the glyoxalate shunt for energy production. However, although the
host manages to control the replication of the M. tb bacilli, the bacteria still survive
and can remain viable for the remaining lifespan of the infected TB patient. The
host response to these persisting bacteria is to form an organised collection of cells
that surround and wall off the microbes in a hypoxic environment where they are
commonly found within lipid rich foamy Ms, surrounded by lymphocytes, giant
cells and fibroblasts. This is called a granuloma (or a tubercle in clinical terminol-
ogy) and contains the TB bacilli, preventing their spread and replication. However,
M. tb within a granuloma are not necessarily eradicated and in many cases can
remain in a dormant physiological state for decades until some extrinsic factor results
in diminished immune regulation of the granuloma that then facilitates reactivation
of bacterial replication, a breakdown of the granuloma’s integrity and breakthrough
16 Mycobacterium tuberculosis Hsp60 as a Key Virulence Factor in Tuberculosis 245

into the airways of the lung. At this point the patient is said to be productive and
will cough up these bacteria which, if inhaled by a new host, complete the infectious
cycle.
Mycobacterium tuberculosis has obviously evolved strategies for evading and
subverting the antimicrobial effector mechanisms of the host immune response. The
prevalence of TB demonstrates how successful these strategies are. However, only
10 % of immunocompetent individuals infected with M. tb develop clinical disease
over their lifetime. Within the other 90 % of infected individuals, the bacterium
survives in a dormant, yet viable state for decades. These data indicate that there
is an ongoing conflict between pathogen and host with the balance being tipped
in the favour of fulminating bacterial disease in some cases, whereas usually the
host can keep the pathogen in check. Which way the balance tips in any given
individual is greatly affected by environmental factors (Lienhardt 2001; van der Eijk
et al. 2007), but also by genetic variation in host susceptibility (Newport and Levin
1999; Doffinger et al. 2006; Stein 2011) and the virulence of the infecting M. tb
isolate (Collins and Smith 1969; North and Izzo 1993; Orme 1999; Sassetti and
Rubin 2003). It has long been known that the virulence (defined as the ability to
produce a progressive infection) of separate strains or isolates of M. tb can vary
in animal models of tuberculosis (Steenken et al. 1934; Alsaadi and Smith 1973;
Orme 1999). Recently, members of the so called Beijing family of M. tb strains have
been shown to have greater infectivity and virulence in man and have rapidly spread
around the world (Bifani et al. 2002; Lasunskaia et al. 2010). It is therefore logical to
propose that specific bacterial genes are critical for the survival and virulence of M. tb
within the host and that identification of these virulence genes and their products will
facilitate the design of novel vaccines and therapies to treat tuberculosis by providing
novel targets for pharmacological research. This has resulted in a concerted effort
within the research community to identify M. tb virulence factors using a variety
of methodologies (Braunstein et al. 2002; Smith 2003; Sharma and Tyagi 2007).
Surveying a transposon mutant library for survival in M (Rengarajan et al. 2005)
or in vivo (Sassetti and Rubin 2003) has identified gene sets that appear to be required
for survival under these experimental conditions which could therefore be considered
the essential genes for virulence. However, alternate methodologies have identified
other gene sets that appear to be virulence factors for the survival of M. tb in M
(Schnappinger et al. 2003; Li et al. 2008; Li et al. 2010) and in vivo (Talaat et al.
2004; Lamichhane et al. 2005). A direct comparison of the gene sets identified to be
virulence factors in three separate M infection studies showed only limited overlap
(Li et al. 2010). Undoubtedly, differences in methodologies account for some of
this variation but different interpretations of what defines a virulence factor should
also be taken into account. Whether it is defined as a genomic difference between a
virulent and an avirulent strain of M. tb or as an expression difference between broth
grown and intracellular bacteria or between intracellular strains of varying virulence,
the identification of a gene product as a virulence factor is open to interpretation.
Although an understanding of the molecular details controlling the pathogenesis of
TB is by no means complete, recent research has begun to determine the microbiology
and immunology of this phenomenon (Glickman and Jacobs 2001; Smith 2003;
246 R. W. Stokes

de Chastellier 2009; Barry et al. 2009; Stokes and Waddell 2009). Mycobacterium
tuberculosis bacilli within their host encounter numerous stresses including residence
within an intracellular environment, exposure to M killing mechanisms, exposure
to the effector mechanisms of the host’s adaptive immune response and changes
to oxygen and nutrient availability (Ehrt and Schnappinger 2009). The pathogen
responds to these stresses in several ways (Ehrt and Schnappinger 2009; Stokes
and Waddell 2009) including the induction of stress proteins of which the Hsp60
homologue is perhaps the best known.

16.2 The Chaperonin 60 (Hsp60/Hsp65/Cpn60) Proteins of

Mycobacterium tuberculosis

The mycobacterial Hsp60 homologue (often called Hsp65 in mycobacteria) was first
identified as a member of the “common antigen” family (Thole et al. 1988a) and
an immunodominant antigen in TB patients and in experimental animal infections
(Young et al. 1988;Young 1990). That the Hsp60 of M. tb is a strong immunogen is not
surprising as members of the Hsp60 family are major antigens in several pathogens
and strong antibody responses to Hsp60 are to be found following bacterial, pro-
tozoan and helminth infections (Young 1990). Early studies on the mycobacterial
Hsp60 included the identification of B cell and T cell epitopes, identifying numerous
epitopes of varying degrees of species specificity and species cross-reactivity (An-
derson et al. 1988; Thole et al. 1988b). It was shown that M. tb Hsp60 could stimulate
T-cell responses in human subjects, irrespective of whether they were infected with
M. tb (Lamb et al. 1986; Thole et al. 1988a). This raised the possibility that recogni-
tion of conserved epitopes in the Hsp60 family could lead to autoimmunity (Lamb
et al. 1989; Dubaniewicz 2010).
Studies have shown that experimental adjuvant arthritis in rats and mice (a model
of rheumatoid arthritis (RA) in humans) can be induced by the injection of intact
mycobacteria or complete Freund’s adjuvant (a mixture of M. tb components within a
mineral oil vehicle) (McLean et al. 1990; Cohen 1991). In contrast, pre-immunization
with recombinant Hsp60 or virally expressed Hsp60 leads to suppression of and/or
protection from adjuvant arthritis (Billingham et al. 1990; Yang et al. 1990; Lopez-
Guerrero et al. 1994; Haque et al. 1996). An explanation for how M. tb Hsp60
induces autoimmunity has not been determined but it appears that repeated exposure
to bacteria, especially pathogens containing proteins with a high similarity to host
mammalian antigens, affects the host’s ability to discriminate between self and non-
self antigens (Moudgil and Sercarz 1994). As mycobacterial and mammalian Hsp60
homologues share 60 % homology (Jindal et al. 1989) and mycobacterial infections
are commonly chronic and may remain with the patient all their lives, it can be
seen why mycobacterial Hsps are commonly implicated in autoimmune diseases.
The autoimmunity induced by mycobacterial Hsp60 could merely be an unavoidable
consequence of the homology seen between Hsps of all species and is of no advantage
to host or pathogen. However, the possibility that the autoimmunity is an occasional
16 Mycobacterium tuberculosis Hsp60 as a Key Virulence Factor in Tuberculosis 247

“by product” of an M. tb virulence strategy should not be discounted. As will be

shown below, Hsp60 of M. tb appears to be actively transported to the outer layers of
the bacterial cell wall and beyond where it interacts with the host. The data showing
that the extracellular Hsp60 is acting as an immunomodulator, a bacterial ligand
mediating attachment to Ms and a cell signaling molecule indicates that the Hsp60
of M. tb has a role in the pathogenesis of TB.

16.2.1 Mycobacterial Paralogues of Hsp60

Most bacteria contain a single Hsp60 gene but it is becoming clear that in approx-
imately 30 % of the bacteria that have been currently sequenced, multiple copies
of Hsp60 exist (Lund 2009). This is true for M. leprae (Rinke de Wit et al. 1992),
M. tb (Kong et al. 1993), M. bovis (Wang et al. 2011) and M. avium paratuberculo-
sis (Goyal et al. 2006) which all have two copies of Hsp60, whereas M. smegmatis
has three copies (Rao and Lund 2010). Phylogenetic analysis suggests that the two
paralogues resulted from a single gene duplication event followed by varied rates
of evolutionary change (Hughes 1993) with the third homologue in M. smegmatis
appearing to have been acquired by horizontal gene transfer (Rao and Lund 2010).
The two paralogues of Hsp60 in pathogenic mycobacteria are designated Cpn60.1
(GroEL1, M. tb genome accession number Rv3417c) and Cpn60.2 (GroEL2, Hsp65,
M. tb genome accession number Rv0440). The Cpn60.1 and Cpn60.2 proteins from
M. tuberculosis only share 61 % sequence identity (Kong et al. 1993) while there
is 95 % identity between Cpn60.2 of M. tb and M. leprae (Shinnick et al. 1987).
This implies that Cpn60.1 and Cpn60.2 would have divergent functions (Qamra et al.
2005). Comparable to the GroEL function in E.coli, Cpn60.2 shows hydrophobicity-
based protein folding activity and is acting as a “normal” chaperonin. However, this
function seems to result from the formation of a Cpn60.2 homodimer that is less
ATP-dependent than is the GroEL of E. coli (Qamra et al. 2004; Shahar et al. 2011).
Both Cpn60.1 and Cpn60.2 behave as dimers in vivo and in vitro which is unlike other
bacterial Hsp60s that exist as tetradecamers (Qamra et al. 2004; Shahar et al. 2011).
As it appears that M.tb Cpn60.2 is acting as a GroEL equivalent, it was surprising
to find that cpn60.1 appears to be arranged in a putative operon with cpn10 (GroES,
M. tb genome accession number Rv3418c) (Kong et al. 1993), while cpn60.2 is
found elsewhere on the chromosome. However, recent studies show that the apical
domains of M. tb Cpn60.1 and Cpn60.2 are conserved in their 3-D structure and
appear to be like the E. coli GroEL. Thus, it seems that while Cpn60.2 functions
as the general housekeeping chaperonin, Cpn60.1, like Cpn60.2, can also act as a
chaperonin (Sielaff et al. 2010), although this is only based on structural homology.
Further support for the divergent functions of Cpn60.1 and Cpn60.2 came from the
attempts to delete these genes in mycobacteria. It was found that a knockout mutant
can be obtained for cpn60.1 in both M. tb (Hu et al. 2008) and M. bovis BCG (Wang
et al. 2011). In contrast, cpn60.2 can not be deleted and has been shown to be an
essential gene required for the survival of M. tb (Hu et al. 2008). The fact that cpn60.2
248 R. W. Stokes

is essential lends support to the idea that it acts as the main housekeeping chaperone
for M.tb in much the same way as GroEL does in E. coli. The role of Cpn60.1 is less
clear. While it can possibly act as a chaperonin (Sielaff et al. 2010), deletion of the
gene in M. tb did not result in a dramatic phenotype (Hu et al. 2008) suggesting that
any chaperonin activity is not essential to bacterial survival. Growth of the mutant
in broth and in Ms was found to be equal to that of the wild-type parent (Hu et al.
2008). However, the mutant failed to grow in mice as rapidly as did the wild type,
attaining comparable bacterial load in the lung and spleen only at later time points.
This was associated with differences in the granulomatous inflammation in both mice
and guinea pigs, with the mutant infected lungs showing only minimal inflammation
in mice at 15 weeks post-infection, even though bacterial numbers were similar to
that of the wild type (Hu et al. 2008). This suggested that Cpn60.1 is essential for
the induction of normal granuloma formation during M.tb infection. The finding that
levels of TNFα, IFNγ, IL-6 and IL-12 in the lungs of mice infected with the mutant
were significantly lower than that seen in mice infected with wild-type bacteria up
to 15 weeks post-infection (Hu et al. 2008) suggests that inflammation is affected
throughout the course of the infection and indicates that Cpn60.1 is important in the
induction of this inflammation. It may seem counter-intuitive that a putative M.tb
virulence factor would induce inflammation, a host response usually associated with
defence against bacterial infection. However, it is important to note that M.tb has a
level of resistance to the effector arm of cell-mediated immunity and, in fact, resides
within the very cells that are part of this response.
The growth of a M. bovis BCG Cpn60.1 mutant in broth was equal to that of the
wild type although more protein was secreted into the supernatant by the mutant
(Wang et al. 2011). Cell wall lipids were altered in the mutant and it was more
susceptible to hydrogen peroxide (Wang et al. 2011). When growth in mice was
investigated, the mutant was slightly less persistent in the lungs and spleen but
retained its ability to protect vaccinated mice against a challenge with M. tb (Wang
et al. 2011). Thus, like M.tb, the growth of a M. bovis BCG Cpn60.1 mutant is not
greatly affected. However, it was shown for BCG that Cpn60.1 was necessary for
bacterial cell wall integrity and resistance to hydrogen peroxide, but is not essential
for the vaccine potential of BCG.

16.3 Secretion of Hsp60

It can thus be seen that the two Hsp60 paralogues in M.tb differ in their essential-
ity for bacterial survival and also in their function. That these stress proteins have
other roles besides that of acting as a chaperone is becoming clear, with increasing
evidence that they are secreted signaling molecules, modulators of host immunity,
surface located bacterial ligands and bacterial cell wall components. A useful term
for these additional roles of M.tb Hsp60 has been suggested by Henderson and his
colleagues (Cehovin et al. 2010; Henderson et al. 2010) who call them “moonlight-
ing” functions—a term initially introduced by Connie Jeffery (Jeffery 1999). Some
16 Mycobacterium tuberculosis Hsp60 as a Key Virulence Factor in Tuberculosis 249

resistance to this idea that stress proteins may have other functions besides acting as
chaperonins has been forthcoming and seems, at least in part, to be connected to the
dogma that M.tb Hsp60 acts only as a chaperonin and is therefore located intracellu-
larly where they can function to mediate protein folding and does not transfer across
the plasma membrane. In fact, it is commonly believed that detection of Hsp60 in
a culture supernatant is indicative of cell lysis (Sonnenberg and Belisle 1997). It is
therefore worthwhile examining the evidence that both Cpn60.1 and Cpn60.2 are
normally to be found both within the cytosol and on the outer layers of the cell wall
where they can be shed or actively secreted into the extracellular environment.
With the demonstration that mycobacteria have multiple copies of Cpn60 and
the increasing demonstrations that Cpn60 has “moonlighting” functions, it becomes
easier to accept that Cpn60 may have functional roles that involve its location other
than in the cytosol. Indeed, Cpn60 has been shown to be secreted and to be located
within the outer layers of the cell wall of M.tb. The identification of M receptors
that mediate binding of intact mycobacteria via Cpn60.2 (Hickey et al. 2009, 2010)
necessitates that the Cpn60.2 must be located at the cell surface of the mycobacteria.
In addition to this evidence, it has been demonstrated that mycobacteria do contain
several Hsps, including Cpn60, within their outer cell wall by using various method-
ologies such as electron microscopy (Esaguy and Aguas 1997), antibody binding
(Gillis et al. 1985; Esaguy and Aguas 1997; Hickey et al. 2009) and proteomics
(Stokes, unpublished observations and (Rosenkrands et al. 2000; Wolfe et al. 2010)).
In fact, using isobaric tags for relative and absolute quantitation (iTRAQ), Cpn60.1
and Cpn60.2, along with Hsp70, Hsp10 and Hsp16 have been shown to be among the
most prevalent of proteins within the outer cell wall capsular layer of M.tb (Stokes
unpublished observations). This is further supported by studies analyzing proteins in
the cell wall of M.tb (Wolfe et al. 2010), by demonstrating the presence of Hsp16 in
the cell wall (Cunningham and Spreadbury 1998) and by protein gel analysis of M.tb
capsule (Hickey et al. 2009). Furthermore, Cpn60.1 has been shown to be secreted
by M.tb into the supernatant of broth cultures (Cehovin et al. 2010). Interestingly,
at the same time point that Cpn60.1 first appears in culture filtrates (6 days), no
Cpn60.2 can be found (Hickey et al. 2009; Cehovin et al. 2010) even though it is on
the surface of the bacteria (Hickey et al. 2009). This would imply that Cpn60.1 is
actively secreted, perhaps to facilitate its actions on host cells. However, it is worth
noting that Cpn60.2 secretion (or release) can be induced by the removal of zinc
from the culture medium (De Bruyn et al. 1989).
To date no mechanism for the active secretion of Cpn60.1 has been identified, nor
has a mechanism for how Cpn60.2 and the other Hsps access their outer cell wall
location been discovered. The means by which these, and, for that matter, the many
other cell wall-located and secreted proteins exit the mycobacterial cytosol are poorly
understood. Although significant progress has been made in identifying the protein
secretion systems of mycobacteria (Abdallah et al. 2007; Digiuseppe Champion and
Cox 2007), none of the systems identified appear (at least, as yet) to be involved in
the transport of Cpn60 across the plasma membrane. However, possible mechanisms
for the egress of Cpn60 and other Hsps can be postulated. For instance, secretion
may be due to their hydrophobic surfaces allowing them to interact with membrane
250 R. W. Stokes

phospholipids and other lipidic molecules within the largely hydrophobic milieu of
the lipid rich mycobacterial cell wall, as suggested for other bacteria (Hennequin
et al. 2001). Indeed, one report has shown that GroEL, human Hsp70, Cpn60.2
and DnaK all have the capacity to induce the formation of pores in lipid bilayers
(Alder et al. 1990). Additionally, GroEL can promote lipid bilayer stability during
protein folding activity (Torok et al. 1997), indicating its ability to traverse the plasma
membrane. Alternatively, Cpn60 may engage more specific export mechanisms such
as ‘hitch-hiker’-based export via the recently described mycobacterial Twin-Arginine
Translocation (Tat) pathway (McDonough et al. 2005; Lee et al. 2006). Proof-of-
principal for the secretion of Hsps exists, even though they have not been specifically
applied to mycobacterial Hsp60. For example, M. tb Cpn10 protein appears to be
secreted from the bacterium, and shares some structural elements common to the
N-terminal region of Hsp60 (Hughes 1993). In addition, the active secretion of
mammalian Hsp60 (Merendino et al. 2010) and Hsp70 (Mambula et al. 2007, see
Chap. 6) demonstrate that Hsp60 could be secreted to the mycobacterial cell surface
and beyond.

16.4 Moonlighting Functions of Bacterial Hsp60 Proteins

Once Hsp60 has traversed the plasma membrane and lipidic layers of the mycobacte-
rial cell wall, what functions does it have? The growing literature on this topic would
suggest several functions. The immunomodulatory function of Cpn60 in autoim-
munity has already been covered above, but other cell-cell signaling mechanisms
for Cpn60 have also been discovered. Hsp60 of the oral bacterium Aggregatibacter
actinomycetemcomitans stimulates osteoclast function resulting in the breakdown of
murine calvarial bone (Kirby et al. 1995; Henderson et al. 2003). Interestingly, the
Hsp60 proteins from both humans and some other bacteria (e.g. E.coli) also have
this function (Reddi et al. 1998; Meghji et al. 2003). However, this shared function
in Hsp60 homologues is not repeated with M.tb. While M. tb Cpn10 (Meghji et al.
1997) has this activity, M. tb Cpn60.2 does not and M. tb Cpn60.1 actually inhibits
osteoclast function (Winrow et al. 2008). Whether these observations are related in
any way to the virulence of intracellular M.tb is not yet clear.
More obviously connected to the virulence of M.tb is the effect of Hsp60 on the
induction of host cell M production of cytokines, reactive oxygen intermediates
(ROI) and reactive nitrogen intermediates (RNI). Early studies did not differenti-
ate the two Hsp60 paralogues but still showed that Hsp60 of M. tb (actually the
Hsp65 or Cpn60.2 protein) induced the production of TNFα, IL-6 and IL-8 by the
human macrophage-like cell line, THP-1 (Friedland et al. 1993) and TNFα and IL-
6 by murine peritoneal Ms (Peetermans et al. 1995). Interestingly, murine Ms
also produced RNI in response to Hsp60 which was TNFα-dependent and inhibited
intracellular replication of the protozoan pathogen, Toxoplasma gondii (Peetermans
et al. 1995). However, whether RNI play any role in human Ms is still a topic of
some controversy (Fang 2004). Hsp60 treatment of human monocyte-derived Ms
16 Mycobacterium tuberculosis Hsp60 as a Key Virulence Factor in Tuberculosis 251

induced the pro-inflammatory cytokines TNFα and IL-1β and increased the expres-
sion of the surface complement receptor 3, but did not result in increased induction
of reactive oxygen intermediates or MHCII expression, indicating a lack of classical
M activation (Peetermans et al. 1994). As it is classical IFNγ -mediated activation
that is able to conrol M.tb intracellular replication (Cooper and Flynn 1995; Doffin-
ger et al. 2006), it would not be advantageous to M.tb to induce this response, while
an increase in complement receptor 3 expression may aid the uptake of the bacteria
in an advantageous manner (Stokes et al. 1993; Velasco-Velazquez et al. 2003).
Following the discovery that M.tb has two paralogues of Hsp60, it was possible to
compare the ability of M. tb Cpn60.1 and Cpn60.2 to induce cytokine production by
Ms. While both Cpn60.1 and Cpn60.2 stimulate human M to produce IL-1, IL-6,
IL-8, IL-10, IL-12, TNFα and GM-CSF but not IL-4 or IFNγ , 100 fold less Cpn60.1
was required to stimulate comparable amounts of these cytokines (Lewthwaite et al.
2001). Furthermore, Cpn60.1 but not Cpn60.2, signalling was shown to involve
CD14 (Lewthwaite et al. 2001). Both Cpn60.1 and Cpn60.2 have only a partial
requirement for MyD88 to induce M cytokine production. Additionally, both have
a requirement for Toll-like Receptor (TLR)-4, with Cpn60.2 having an additional
requirement for TLR2 (Cehovin et al. 2010). Additional studies showed that both
Cpn60.1 and Cpn60.2 utilize the ERK/1 and MAPK signaling pathways to induce
cytokine production by M (Lewthwaite et al. 2007). When whole blood leucocyte
populations are stimulated with Cpn60.1 and Cpn60.2, only IL-1β and IL-6 and not
IL-8, IL-10, IL-12 or IFNγ were produced by the mixed cell population. In this
model, Cpn60.2 was a more potent stimulator than was Cpn60.1 and was the only
one that induced TNFα production (Cehovin et al. 2010). The contrasting results
with those previously reported for M (Lewthwaite et al. 2001) indicated that the
interaction of Cpn60.1 and Cpn60.2 with whole blood was very different from that
seen with pure M.

16.5 Binding of Hsp60 to Immune Effector Cells

The demonstration that TLRs, CD14 and MyD88 are necessary for appropriate sig-
naling to take place in response to Cpn60 does not mean that they are necessarily the
receptors for Cpn60. In another model studying the interaction of lipopolysaccha-
ride (LPS) with M, it was shown that CD14, TLR4, MD2 and other cell surface
moieties form an intricate complex that mediates binding and cell signaling in re-
sponse to LPS (Triantafilou and Triantafilou 2005, see also Chap. 10). Perhaps a
similar complex of M surface receptors is needed to interact with mycobacterial
cell wall glycolipids and proteins. Nevertheless, the search for M receptors that
bind Cpn60 has indicated a number of cell surface proteins that may bind to Cpn60.1
(Henderson and Mesher 2007). Binding of Hsp70 and Cpn60.1 from M. bovis BCG
to DC-SIGN has also been reported (Carroll et al. 2010). In contrast, it appears
that, in the absence of serum (a situation that would be found within the alveolar
space where M.tb first encounters Ms), Cpn60.2 binds strongly to the M surface
252 R. W. Stokes

receptor, CD43 (sialophorin, leukosialin) and that this receptor/ligand interaction ac-
counts for 30–40 % of all binding of M. tb bacilli to Ms (Hickey et al. 2009, 2010).
Whether this binding can be considered a true receptor/ligand interaction and not just
an interaction of “sticky” chaperonins with a host glycoprotein is not unequivocally
determined. However, two observations strongly suggest that this interaction is a
specific binding of the two moieties: (i) although both Cpn60.1 and Cpn60.2 are
present in large amounts in the outer cell wall capsule of M.tb (Stokes, unpublished
data), only Cpn60.2 binds to isolated CD43 (Hickey et al. 2009) and (ii) Hsp70 was
also shown to bind to isolated CD43 but does not mediate binding of whole bacteria
to CD43 on Ms (Hickey et al. 2009, 2010). It is interesting to note that Cpn60.1
was found to bind to approximately 90 % of circulating human monocytes compared
to <50 % binding with Cpn60.2 (Cehovin et al. 2010). This may reflect the very dif-
ferent surface receptors found on monocytes and M, although CD43 is expressed
on both.
The finding that Cpn60.2 can interact with purified CD43 (Hickey et al. 2009,
2010), does not necessarily mean that they interact with M surface CD43 in iso-
lation. It is possible that CD43 interacts with mycobacteria within the context of a
group of M surface molecules, as was described above for LPS (Triantafilou and
Triantafilou 2005, see Chap. 10). In this model, CD43 would co-operate with other
surface M receptors to facilitate efficient bacterial binding and/or signal transduc-
tion via interaction with one or more bacterial surface molecules. The demonstration
that soluble CD43 can overcome the deficiency of mycobacterial binding to M
from CD43 knockout mice (Fratazzi et al. 2000) suggests that, although M. tb can
bind CD43 directly, it may also interact with other M receptors. In addition to
Cpn60.2, numerous other mycobacterial cell wall constituents have been identified
as ligands that mediate binding to M and several M receptors have been shown
to be involved in this binding (El-Etr and Cirillo 2001; Schafer et al. 2009; Mishra
et al. 2011). Alternatively, it may be that Cpn60.2 and CD43 do interact in isolation
and that this interaction anchors the M.tb, thereby facilitating subsequent ligand-
receptor interactions to effectively take place such as binding by the phagocytic CR3
receptor (Melo et al. 2000; Rooyakkers and Stokes 2005), or signaling via TLRs
(Means et al. 1999; Thoma-Uszynski et al. 2001; Reiling et al. 2008). It is note-
worthy here that CD43 often plays the role of an intercellular binding modulator,
allowing some receptor-ligand interactions to take place more readily, while limiting
other interactions (Ostberg et al. 1998).
Whether this interaction of CD43 with M.tb Cpn60.2 is to the advantage of the
bacteria or the host is not clear yet. It is known that absence of CD43 results in
more rapid bacterial growth in Ms and a more severe pathology resulting from
M.tb infection in vivo (Randhawa et al. 2005). Increased growth of M.tb in CD43
null Ms is due to a reduction in TNFα -mediated apoptosis of these Ms that then
allows for greater bacterial replication (Randhawa et al. 2008). This would suggest
that recognition of Cpn60.2 by CD43 is a host defence mechanism and not a bacterial
virulence strategy. Recognition of an essential M.tb protein that results in induction
of a mechanism to control the intracellular replication of the pathogen would be a
good defence strategy for the host. As M.tb can not survive without Cpn60.2, it has
16 Mycobacterium tuberculosis Hsp60 as a Key Virulence Factor in Tuberculosis 253

little opportunity to avoid this immune defence mechanism. However, whether the
induction of TNFα-mediated apoptosis via CD43 is facilitated by Cpn60.2 or some
other M.tb surface moiety binding to the CD43 is not yet unequivocally determined.
Another intriguing possibility is that the role of secreted Cpn60.1 may be to counter
the host defence mechanisms initiated by recognition of Cpn60.2, thus providing
one possible explanation for the evolution of two Cpn60 paralogues in M.tb.
Genetic evolution analyses provide evidence that an ancient mycobacterial an-
cestor gained an additional Cpn60 copy at some point and since that time Cpn60.1
has undergone a more rapid level of nonsynonomous mutation, apparently leading
to a form that no longer functions in protein folding, while Cpn60.2 has evolved
to facilitate protein folding without the need of Cpn10 (Hughes 1993; Qamra et al.
2004). That GroE (GroEL + GroES) is necessary for the formation and mainte-
nance of the E. coli cell wall suggests that these chaperonins may have originally
located to the cell wall to facilitate cell maintenance (McLennan and Masters 1998).
In addition, Cpn60.1 from M. smegmatis has been implicated in the formation of
mycolic acids, again suggesting a functional role within the mycobacterial cell wall
(Ojha et al. 2005). These observations suggest that at least one reason that bacterial
molecular chaperones leave the cytosolic space is to facilitate their role in main-
tenance of the cell wall. Thus, the additional “moonlighting” roles that molecular
chaperones demonstrate may have evolved as a byproduct of this extracellular lo-
calization. An additional means by which the Cpn60 proteins may have attained
additional functions relates to the fact that the mycobacteria contain multiple copies
of these proteins. The finding that only Cpn60.2 is necessary for viability suggests
that Cpn60.1 and Cpn60.2 have unique roles within the bacterium (Hu et al. 2008)
and that Hsps can evolve to have additional functions if another functional copy is
retained for housekeeping functions related to cellular viability (Hu et al. 2008).

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