Moonlighting Actions of Mycobacterial Chaperonin 60 Proteins
One of the earliest studies that revealed that bacterial and eukaryotic cell stress proteins had cell signaling actions that could render them pro- and/or antiinflammatory proteins (Henderson and Pockley 2010) was that the M. tuberculosis chaperonin 60.2 (HSP65) protein was able to stimulate the human monocyte cell line THP-1 to generate the key early-response proinflammatory cytokine, TNFa (Friedland et al. 1993). This finding could either be interpreted as the recombinant protein being contaminated with the major Gram-negative bacterial component, lipopolysaccharide (LPS), or that this protein had actions similar to LPS and was able to induce macrophages to become classically activated. This is similar to the M1 state mentioned above (Martinez and Gordon 2014). This classic activation state is presumed to have evolved to allow macrophages to kill microbes and present microbial antigens to T lymphocytes. This involves multiple changes in the macrophage including upregulation of expression of Fc receptors, MHC class II, and production of oxygen-derived free radicals (Fujiwara and Kobayashi 2005). It would therefore appear that M. tuberculosis chaperonin 60.2 is able to induce classic macrophage activation and is part of the process of induction of immunity to M. tuberculosis. However, a separate study of this protein found that it did not promote this panoply of changes in macrophages, but only induced the synthesis of cytokines (Peetermans et al. 1994). This suggests that M. tuberculosis chaperonin 60.2 is inducing a state of alternative activation in these macrophages.
Inflammation involves the trafficking of blood monocytes to sites of bacterial infection. To cross the vascular endothelial barrier, leukocytes need to interact with a range of adhesive proteins on vascular endothelial cells (VECs) including E-selectin and members of the ICAM and VCAM families. These proteins are normally inducible either by LPS or by the proinflammatory cytokines such as IL-1 and TNF - with the LPS being responsible for the synthesis of these cytokines by the VEC - which then acts in a paracrine/autocrine manner (Koizumi et al. 2003). Mycobacterium tuberculosis chaperonin 60.2 was, like LPS, also able to induce the expression of E-selectin, ICAM-1, and VCAM-1 on cultured human VECs, and to promote the adhesion of human leukocytes to these cells. However, unlike LPS, this induction of adhesion is a direct effect of the chaperonin 60.2 protein, and was not associated with the synthesis of IL-1 or TNF (Verdegaal et al. 1996). The literature reveals that only a small number of bacterial components are able to activate VEC adhesion and this is due to the promotion of cytokine synthesis. This direct activity of M. tuberculosis chaper- onin 60.2 on human VECs therefore appears to be a unique effect, which is likely to be of significant importance provided that the chaperonin 60.2 protein is secreted. Indeed, as will be described, the chaperonin 60.2 protein of M. tuberculosis is secreted and can function as a cell-wall protein (Hickey et al. 2009, 2010). Moreover, secretion can be modulated, as in Mycobacterium bovis; the inactivation of the gene encoding chaperonin 60.1 increases, by an unknown mechanism, the secretion of the chaperonin 60.2 protein 200-fold (Wang et al. 2011). In addition, a cell-surface serine hydrolase of M. tuberculosis known as Hip1 (hydrolase important for pathogenesis 1) was found to be a modulator of innate immunity and functioned to control the release of chaperonin 60.2 (Rengarajan et al. 2008). Further work has identified that cleavage of M. tuberculosis chaperonin 60.2 by Hip1 at position 18 in the N-terminus produces a protein with no oligomeric structure and with significantly less proinflammatory activity than the native protein, so decreasing the host immune response to the bacterium (Naffin-Olivos et al. 2014). Interestingly, the nitric oxide-responsive transcription factor WhiB1inhibits transcription from the chaperonin 60.2 promoter, blocking the action of the positively acting CRP family transcription factor Cmr (Stapleton et al. 2012).
With the discovery of the second chaperonin 60 gene in M. tuberculosis, the obvious question was whether this protein also had proinflammatory activity. The first activity that both proteins were compared for was bone resorption. This arose from the author's finding that the chaperonin 60 protein of E. coli was a potent stimulator of bone resorption, which worked by inducing the formation of the multinucleated myeloid cells (known as osteoclasts) which control the normal turnover of bone (Kirby et al. 1995; Reddi et al. 1998). Surprisingly, both chaperonin 60 proteins of M. tuberculosis had no stimulatory effect in the assay used to detect agents able to promote bone destruction (resorption) (Meghji et al. 1997). This was one of the first studies to show that moonlighting proteins within a protein family (in this case the chaperonin 60 family of proteins) could have distinct biological actions.