The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8(+) T Cells

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+) and CD8(+) T cells secreting gamma interferon (IFN-gamma) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

Molecular Mechanism Underlying ATP-Induced Conformational Changes in the Nucleoprotein Filament of Mycobacterium smegmatis RecA

RecA plays a central role in bacterial DNA repair, homologous recombination, and restoration of stalled replication forks by virtue of its active extended nucleoprotein filament. Binding of ATP and its subsequent recognition by the carboxamide group of a highly conserved glutamine (GIn196 in MsRecA) have been implicated in the formation of active RecA nucleoprotein filaments. Although the mechanism of ATP-dependent structural transitions in RecA has been proposed on the basis of low-resolution electron microscopic reconstructions, the precise sequence of events that constitute these transitions is poorly understood. On the basis of biochemical and crystallographic analyses of MsRecA variants carrying mutations in highly conserved Gln196 and Arg198 residues, we propose that the disposition of the interprotomer interface is the structural basis of allosteric activation of RecA. Furthermore, this study accounts, for the contributions of several conserved amino acids to ATP hydrolysis and to the transition from collapsed to extended filament forms in Mycobacterium smegmatis RecA (MsRecA). In addition to their role in the inactive compressed state, the study reveals a role for GIn196 and Arg198 along with Phe219 in ATP hydrolysis in the active extended nucleoprotein filament. Finally, our data suggest that the primary, but not secondary, nucleotide binding site in MsRecA isomerizes into the ATP binding site present in the extended nucleoprotein filament.