Systematic Evaluation of Mycobacterium tuberculosis Proteins for Antigenic Properties Identifies Rv1485 and Rv1705c as Potential Protective Subunit Vaccine Candidates.

The lack of efficacious vaccines against Mycobacterium tuberculosis (MTB) infection is a limiting factor in the prevention and control of tuberculosis (TB), the leading cause of death from an infectious agent. Improvement or replacement of the BCG vaccine with one that reliably protects all age groups is urgent. Concerns exist that antigens currently being evaluated are too homogeneous. To identify new protective antigens, we screened 1,781 proteins from a high-throughput proteome-wide protein purification study for antigenic activity. Forty-nine antigens (34 previously unreported) induced antigen-specific gamma interferon (IFN-γ) release from peripheral blood mononuclear cells (PBMCs) derived from 4,452 TB and suspected TB patients and 167 healthy donors. Three (Rv1485, Rv1705c, and Rv1802) of the 20 antigens evaluated in a BALB/c mouse challenge model showed protective efficacy, reducing lung CFU counts by 66.2%, 75.8%, and 60%, respectively. Evaluation of IgG2a/IgG1 ratios and cytokine release indicated that Rv1485 and Rv1705c induce a protective Th1 immune response. Epitope analysis of PE/PPE protein Rv1705c, the strongest candidate, identified a dominant epitope in its extreme N-terminal domain accounting for 90% of its immune response. Systematic preclinical assessment of antigens Rv1485 and Rv1705c is warranted.

Mycobacterium tuberculosis CRISPR/Cas system Csm1 holds clues to the evolutionary relationship between DNA polymerase and cyclase activity.

Prokaryotic CRISPR/Cas systems confer immunity against invading nucleic acids through effector complexes. Csm1, the signature protein of Type III effector complexes, catalyses cyclic oligoadenylate synthesis when in the effector complex, but not when alone, activating the Csm6 nuclease and switching on the antiviral response. Here, we provide biochemical evidence that M. tuberculosis Csm1 (MtbCsm1) has ion-dependent polymerase activity when independent of the effector complex. Structural studies provide supporting evidence that the catalytic core of the MtbCsm1 palm2 domain is almost identical to that of classical DNA polymerase Pol IV, and that the palm1 and B domains function as the other structural elements required (thumb and fingers) for DNA polymerase activity. MtbCsm1 polymerase activity is relatively weak in vitro and its functional relevance in vivo is unknown. Our structural and mutagenesis data suggest that residue K692 in the palm2 domain has been significant in the evolution of Csm1 from a polymerase to a cyclase, and support the notion that the cyclase activity of Csm1 requires the presence of other elements provided by the CRISPR/Cas effector complex. This structural rationale for Csm1 polymerase (alone) and cyclase (within the effector complex) activity should benefit future functional investigations and engineering.