Actions of Mycobacterial Chaperonin 60 Proteins Compatible with the Pathology of Tuberculosis

Since 2000, a small but growing number of studies of the chaperonin 60 proteins of the mycobacteria have revealed their role in the relationship between these bacteria and their host. The two M. tuberculosis chaperonin 60 proteins exhibit 76% amino acid sequence similarity, which would suggest these proteins have identical biological actions. However, the chaperonin 60.1 protein is a significantly more active monocyte-activating ligand than the chaperonin 60.2 protein. This may be related to the fact that chaperonin 60.2 activity is dependent on binding to CD14 while that of chaperonin 60.1 is only partially CD14-depend- ent, suggesting these proteins work via different receptor complexes at the cell surface (or within the cell). This was the first evidence that M. tuberculosis chaperonin 60.1 could have a role in virulence (Lewthwaite et al. 2001).

These findings were of interest, but could be artifacts of the in vitro system being used. However, two separate groups have looked at mycobacterial chaperonin 60 proteins in in vivo models of the human lung disease, asthma. This is a chronic inflammatory lung disease. Gelfand's group examined the chaperonin 60 proteins from Streptococcus pneumonia, Helicobacter pylori, and bacillus Calmette-Guerin (BCG) and from Mycobacterium leprae and M. tuberculosis (both chaperonin 60.2) for their effects in a mouse model of asthma. Only the administration of the M. leprae chaperonin 60.2 protein had therapeutic effects, including inhibition of the Th2 cytokines IL-4 and IL-5 (Rha et al. 2002). It was notable that administration of as little as 10 pg recombinant chaperonin 60 to mice inhibited asthma. In a subsequent study from this group, the in vivo actions of the M. leprae chaperonin 60.2 protein in this model was shown to be due to the ability of this protein to upregulate the expression of the Notch receptor ligand, Delta 1, expression on dendritic cells. This appeared to be associated with the chaperonin 60.2-activated dendritic cells being able to skew CD4 T lymphocytes to a Th1 profile of cytokine production (Shin et al. 2012). Using the same model to compare the activities of the M. tuberculosis chaperonins it was found that the chaperonin 60.1 protein and, to a lesser extent, the chaperonin 10 protein had therapeutic activity, but the chaperonin 60.2 protein had no influence on this experimental lung inflammation model (confirming Gelfand's findings) (Riffo-Vasquez et al. 2004). This is an unusual finding, given the results from Gelfand's group with the M. leprae chaperonin 60.2 protein. These proteins share 95% sequence identity, suggesting that the active moonlighting site in the M. leprae protein is relatively small and has, essentially, been mutated in the M. tuberculosis chaperonin 60.2 protein to be biologically inactive. Subsequent studies of the mechanism of action of the M. tuberculosis chaperonin 60.1 protein in this model revealed a direct effect of the protein on leukocyte diapedesis which is related to the inhibition of VCAM-1 expression and upregulation of vascular endothelial cadherin expression (Riffo-Vasquez et al. 2012).

These studies reveal that the chaperonin 60 proteins of the mycobacteria have biological activity in the intact animal and, of relevance to tuberculosis, in the lung. Moreover, they reveal that these chaperonin 60 proteins are active at picomolar to nanomolar concentrations, based on the molecular mass of the monomeric subunit. If the active protein is oligomeric, then these proteins are even more active. The difference in activities of the chaperonin 60.1 and 60.2 proteins suggests that only minor changes in protein sequence, which is presumably related to protein structure, can switch on or off this anti-inflammatory activity. Of note, the in vitro studies of the chaperonin 60.1 and 60.2 proteins would have suggested that both proteins were proinflammatory virulence factors.

One of the complexities in biology is that the actions of individual proteins are context-dependent. These in vivo experiments in an asthma model suggest that, where active, the mycobacterial chaperonin 60 proteins can switch off a Th2- mediated inflammation possibly by redirecting lymphocytes into a Th1-type of behavior. However, Mande's group have shown that, in in vitro studies, the M. tuberculosis chaperonin 60.1 protein can inhibit the actions of the proinflammatory M. tuberculosis component known as purified protein derivative (PPD). This appears to be due to the interaction of chaperonin 60.1 with TLR2 and the skewing of lymphocytes to a Th2-type phenotype (Khan et al. 2008).

So far, the actions of the mycobacterial chaperonins seem to be compatible with the hypothesis that these proteins are cellular ligands and signaling molecules able to modulate inflammation - provided that the proteins are released by the bacterium. It has also been established that the M. tuberculosis chaperonin 60.2 protein is present in substantial amounts on the bacterial surface, where it aids in the interaction of the bacterium with the key cell population in tuberculosis: the macrophage (Hickey et al. 2009). This protein functions as another class of bacterial virulence factor: the adhesin. Bacteria adhesins normally bind to a limited number of cell surface or extracellular matrix molecules including fibronectin, various collagens, laminin, and the like. The receptor for M. tuberculosis chaperonin 60.2 is unique to bacterial adhesins, being the cell-surface sialylated glycoprotein CD43 (Hickey et al. 2010). As CD43 also appears to be important in a range of T-cell functions, including regulating CD4 T lymphocyte trafficking (Cannon et al. 2011), it is possible that this chaperonin 60.2/CD43 interaction may have additional roles in controlling granuloma formation. It has recently been reported that common polymorphisms in the CD43 gene region are associated with susceptibility to tuberculosis and disease severity (Campo et al. 2015). The interaction ofM. tuberculosis chaperonin 60.2 with CD43 reveals an important finding that protein moonlighting is able to expand the protein functional repertoire of this bacterium. This use of multifunctional proteins thus allows for greater genetic efficiency.

The two chaperonin 60 proteins of M. tuberculosis appear to have distinct biological actions both in vitro and in vivo, but little is known about the interactions of these proteins with the macrophage cell surface. A recent study has shown that the chaperonin 60.1 protein has different effects on macrophages depending on whether it binds to TLR2 or TLR4 and explains the findings of Mande's group (Khan et al. 2008). When chaperonin 60.1 binds to TLR2 the complex is endocytosed via a clathrin-dependent mechanism, which results in the production of the anti-inflammatory cytokine IL-10. Inhibition of the endo- cytosis of chaperonin 60.1 results in inhibition of IL-10 synthesis and generation of the proinflammatory cytokine, TNFa. In contrast, interaction of chaperonin

60.1 with TLR4 is not associated with significant endocytosis and triggers a proinflammatory response (Parveen et al. 2013). This shows that this one moonlighting protein, depending on its interaction, can cause distinct activation states of macrophages, such as has been proposed to occur in the tuberculoid granuloma (Ramakrishnan 2012).

One of the major problems in defining the actions of bacterial moonlighting proteins is that it is rarely possible to inactivate the genes encoding these proteins, as these genes code for essential proteins. An attempt was made to inactivate the two chaperonin 60 genes and the chaperonin 10 gene in M. tuberculosis (Hu et al. 2008). The chaperonin 10 and chaperonin 60.2 genes could only be inactivated if a plasmid-based copy of the chromosomal inactivated gene was provided, revealing that these proteins were essential and that the chaperonin 60.2 protein was the major 60 kDa cell stress protein in M. tuberculosis. In contrast, mutants lacking the activity of the gene coding for chaperonin 60.1: (1) were viable; (2) grew at the normal rate in culture and within macrophages; and (3) responded like the wild-type organism to a range of stresses. This suggested that the chaperonin

60.1 protein had lost its protein-folding actions, confirmed by the finding that the gene for M. tuberculosis chaperonin 60.2 could replace E. coli GroEL, but this was not possible with the gene for the chaperonin 60.1 protein (Hu et al. 2008). This suggested that the chaperonin 60.1 protein was “surplus to requirement" However, when the chaperonin 60.1 mutant was used to infect mice or guinea pigs it failed to induce a granulomatous inflammatory state in the lungs, even though it grew at the same rate as the wild-type organism. This suggests that the chaperonin 60.1 protein of M. tuberculosis is a key virulence factor in inducing the granulomatous inflammatory state that characterizes tuberculosis. Further evidence for this hypothesis comes from the use of a human blood granuloma assay described earlier in this chapter (Puissegur et al. 2007). Wild-type M. tuberculosis induced the production of multiple multinuclear macrophages from human whole blood. In contrast, the mutant organism lacking the gene for chaperonin 60.1 was virtually unable to generate such cells (Cehovin et al. 2010). Further evidence for a role ofM. tuberculosis chaperonin 60.1 in the multinuclea- tion of myeloid cells has come from a study of the influence of this protein on osteoclast formation. It turns out that this protein is a potent inhibitor of osteoclast formation both in vitro and in vivo. Administration of M. tuberculosis chaperonin 60.1 to rats with adjuvant arthritis therefore fails to inhibit joint inflammation, but virtually abolishes the massive induction of osteoclastogene- sis that occurs in this model of human rheumatoid arthritis (Winrow et al. 2008). The ability to inhibit osteoclast formation is due to the inhibition of transcription of the gene encoding the major osteoclast transcription factor, NFATc1 (Winrow et al. 2008). It would therefore appear that M. tuberculosis chaperonin 60.1 is an inducer of multinucleate giant cell formation, but an inhibitor of another form of multinucleate giant cell: the osteoclast. This protein appears to be an interesting ligand which could be used to investigate the pathways leading from the monocyte to the various multinucleate giant cells of the human body.

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