Infection Biology of a Novel α-Crystallin of Mycobacterium tuberculosis: Acr2

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the α-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Δacr2) was unimpaired in log phase growth and persisted in IFN-γ-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-γ-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.

Increased knowledge of the function and antigenic properties of Mycobacterium tuberculosis (MTB)4 proteins will assist the design of vaccines and immunodiagnostic reagents against tuberculosis, a disease that kills up to 2 million people per year (1). The heat shock proteins of MTB are of particular interest because their elevated expression is required for bacterial adaptation to adverse conditions encountered during infection, while at the same time providing a signal for host immune recognition (2, 3, 4). Previous work in our laboratory showed that constitutive overexpression of Hsp70 and related heat shock proteins as a result of deletion of the HspR transcriptional regulator induced an increased immune response that significantly impaired the ability of MTB to persist during the chronic phase of infection in mice (5). Whole-genome expression analysis of the HspR deletion strain, and heat-shocked wild-type MTB, identified Rv0251c as a novel major heat shock gene (6). This finding has been confirmed and extended by the comprehensive transcriptomic analysis of Schnappinger et al. (7). Annotated as hsp in the MTB H37Rv genome sequence (8), Rv0251c was found to be induced in naive and activated murine macrophages, and by heat shock, H2O2, SDS, high-dose NO, and palmitic acid in broth cultures (7). On this basis, they categorized Rv0251c as belonging to a group of seven MTB genes that are up-regulated in response to multiple stresses.

Rv0251c encodes a protein with 30% amino acid sequence identity to the well-characterized 16-kDa α-crystallin homolog Acr (encoded by Rv2031c, and referred to as acr or hspX) (6, 8). The identity increases to 41% based on comparison of residues present in the α-crystallin core. The acr gene is known to be required for log phase growth of MTB and during the transition to stationary phase, at which time it is suspected to contribute via chaperonin activity to the stabilization of intracellular structures (9, 10, 11). In addition to this important role in bacillary physiology, the protein is also immunodominant in humans (12, 13, 14). In contrast to acr2, the gene for acr is induced by oxygen deprivation or by exposure to low concentrations of NO in vitro, and its induction in murine macrophages is clearly NOS2 dependent (7, 15, 16). We have previously named Rv0251c acr2 because it is the second member of the α-crystallin family to be identified in MTB. From this point in this report we therefore refer to Acr1 and Acr2 (6).

We investigated the biology of acr2 by measurement of its RNA accumulation in human mononuclear phagocytes, phenotypic analysis of a mutant MTB that lacks the acr2 gene (Δacr2), and analysis of immune recognition of the recombinant Acr2 protein. We found that the level of acr2 RNA increases shortly after phagocytosis of MTB in response to exposure to host reactive oxygen intermediates (ROI). However, the Δacr2 strain was only mildly compromised in its ability to resist various in vivo and in vitro stress stimuli. By virtue of its early expression, we found that Acr2 is a dominant immune target of the early bovine and human T cell responses to mycobacterial infection. The results suggest that, although acr2 plays a role in bacillary defense, its early expression evokes a vigorous early immune response. Thus, Acr2 may be a rare Achilles’ heel for MTB, giving away its presence early after infection.