Stress Wars: the Direct Role of Host and Bacterial Molecular Chaperones in Bacterial Infection (2006)

Since 1962, when Ferruccio Ritossa discovered new puffing patterns in the polytene chromosomes of Drosophila incubated at an elevated temperature (153), we have been aware that stress at the cellular level is answered by the production of specific gene products. These products are variously termed heat shock proteins (Hsps) or cell stress proteins and were originally identified as molecules that are produced in response to the presence of unfolded proteins within the cell (2). However, it was not until the pioneering work of the groups of Laskey, Ellis, and Georgopoulos that the relationship between the generation of correctly assembled macromolecules and the proteins that function to ensure correct assembly was established. Laskey and coworkers studied the nuclear protein nucleoplasmin, which ensures correct assembly of histones and DNA into nucleosomes. Laskey termed nucleoplasmin a molecular chaperone as it mimicked the function of a human chaperone who ensures correct interactions between people (98). Ellis and Georgopoulos studied the protein which eventually was known as chaperonin 60 (Cpn60) and which was responsible for initiating the vast flood of papers on molecular chaperones over the past two decades (58). Currently, a molecular chaperone is defined by Ellis as “one of a large and diverse group of proteins that share the property of assisting the non-covalent assembly/disassembly of other macromolecular structures, but which are not permanent components of these structures when they are performing their normal biological functions” (58). A further refinement of terminology is necessary as certain molecular chaperones are produced constitutively and the concentrations within the cell do not increase in response to stress. These proteins are defined as molecular chaperones but are not Hsps or stress proteins. Molecular chaperones, whose concentrations increase in response to stress, are both chaperones and stress proteins/heat shock proteins.

The first protein-folding molecular chaperone to be discovered was Cpn60 (58). Since the identification of this protein as a molecular chaperone in 1988, many more proteins with actual or putative molecular chaperone functions have been discovered, and the term currently applies to 25 families of proteins (Table ​(Table1).1). In all three kingdoms of life molecular chaperones are classified as essential proteins, and there is significant conservation of sequences between proteins used by prokaryotes and proteins used by eukaryotes (such as thioredoxin [Trx] family members, cyclophilins, chaperonins, Hsp70, and Hsp90). Eukaryotic cells have multiple compartments (cytosol, endoplasmic reticulum, mitochondria, nucleus), and in these compartments stress-induced protein folding is known as the unfolded protein response (158). The unfolded protein responses are an important element in the integrated biology of the cell, are linked to key intracellular signaling pathways, and are now being associated with human disease states (158). Implicit in the definition of molecular chaperones was that they were intracellular proteins involved in the folding of client proteins within cellular compartments, which, because of the high protein concentration (on the order of 200 to 400 mg/ml), favor inappropriate protein-protein interactions, resulting in significant protein denaturation (58). However, it is becoming clear that many molecular chaperones can exist outside the cell and participate in nonfolding actions.



TABLE 1.

Eukaryotic and prokaryotic molecular chaperone and stress protein families



Analysis of immune responses to bacteria in the 1970s identified what was termed a “common antigen” in many bacterial species (81). Patients infected with Mycobacterium tuberculosis or Mycobacterium leprae exhibit significant antibody responses to a 65-kDa antigen (189). Subsequent work identified this antigen (and the common antigen) as the molecular chaperone Cpn60 (193). It has now been established that a number of molecular chaperones from bacteria and protozoan parasites (Cpn60, Hsp70, and Hsp90) are (i) potent immunogens, (ii) active immunomodulators, and (iii) inducers of cross-reactive immunity and autoimmunity (176). The mammalian immune system recognizes the molecular chaperones of infecting parasites as particularly strong immunological signals, which is surprising in view of the significant homology between host and parasite proteins. This profound immune responsiveness should be useful in developing vaccines against pathogens. The Cpn60 (Hsp65) protein of M. tuberculosis has also proven to be an extremely powerful immunomodulator that is able to protect against a number of experimental autoimmune diseases in rodents, including diabetes. In a recent phase II study, immunization of individuals who had newly developed type I diabetes with a peptide derived from human Cpn60 proved to be effective in limiting the progression of this disease (146).

Most researchers studying molecular chaperones work within the paradigm that these proteins are present solely in intracellular compartments. However, in 1989, just 1 year after the identification of Cpn60 as a molecular chaperone, Japanese scientists found that the protein-folding catalyst and stress protein thioredoxin was secreted by T cells from patients with a certain form of leukemia and was able to induce T cells to express one of the subunits of the interleukin-2 (IL-2) receptor (167). Subsequently, the human protein was found to be a potent chemoattractant for neutrophils, monocytes, and T lymphocytes with a unique mechanism of action (124). Since this initial discovery, a growing number of mammalian molecular chaperones have been found to be secreted onto the cell surface or into the extracellular milieu, either tissue culture fluid (with cultured cells) or biological fluids such as blood, synovial fluid, or bronchoalveolar secretions (Table ​(Table2).2). Most of these secreted proteins have agonist activity with a mammalian cell population(s), normally myeloid and lymphoid cells and/or vascular endothelial cells (VECs) (62). Although less attention has been paid to the cell-cell signaling activity of bacterial molecular chaperones, bacterial Cpn10, Cpn60, and Hsp70 have been reported to stimulate or inhibit the proinflammatory actions of myeloid cells and VECs (62). Thus, it is becoming clear that molecular chaperones are examples of moonlighting proteins, that is, proteins which have more than one function. The enzymes of glycolysis are the prototypic moonlighting proteins. For example, secreted phosphoglucoisomerase has been identified as three distinct cytokines: neuroleukin, autocrine motility factor, and differentiation and maturation mediator. This protein also acts as an implantation factor (76). Perhaps the most bizarre example of the moonlighting functions of molecular chaperones is the neurotoxin used by a hunting insect, the antlion or doodlebug, to paralyze its prey. This toxin is produced by a symbiotic bacterium, Enterobacter aerogenes, which lives in the insect's saliva. It has been determined that this toxin is a molecular chaperone, Cpn60. The E. aerogenes Cpn60 is almost identical to the Escherichia coli Cpn60 protein GroEL. Surprisingly, single-residue substitutions in GroEL can change it from an inactive protein into a potent insect neurotoxin (187).



TABLE 2.

Molecular chaperones found on the cell surface and/or secreted by cells and/or found in extracellular fluids



In the last decade there have been a number of reports which support the hypothesis that inducible molecular chaperones, produced both by bacteria and by hosts, function as intracellular, cell surface, or extracellular signals which are involved in the control of the infectious process. This suggests that infection, among other things, is a contest of stress mechanisms with a multitude of unexpected evolutionary twists and turns. In this review we focus on bacterial infection but discuss examples from eukaryotic parasites that exemplify particular mechanisms. The molecular chaperones involved in the formation of the type III secretion system, while indirectly contributing to bacterial virulence, are not included here as they have been extensively reviewed elsewhere (48).