Interaction of Mycobacterium tuberculosis cell wall components with the human natural killer cell receptors NKp44 and Toll-like receptor 2.

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll-like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl-arabinogalactan-peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN-γ production at levels comparable to M. bovis Bacillus Calmette-Guérin-stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG-exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN-γ production were inhibited by anti-TLR2 MAb, but not by anti-NKp44 MAb. Differently, anti-NKp44 MAb partially inhibited CD69 expression on NK cells pre-activated with IL-2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR-2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN-γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Purification of recombinant proteins having high isoelectric points.

The use of recombinant antigens and chemically synthesized peptides are the new approaches for the construction of reliable and sensrtive diagnostic assays. Moreover, in the field of virology, the use of recombinant antigens eliminates the need to handle highly hazardous material in the preparation of the assays (1,2) and allows the assembly of multiepitope polypeptides (3). The protein purification is a critical step in the preparation of recombinant antigens. It is important to point out that the biochemical characteristics of a recombinant protein, expressed in a heterologous system, are unique. Purification problems may be very different for related structural proteins expressed in the same host or for the same protein expressed in different hosts. The designing of an antigen purification procedure is strongly dependent on the immunodominant epitopes present on its surface. The antibodies recognize chemical groupings, exposed to the solvent, on the surface of an antigen An immunodominant determinant may be continuous or discontniuous. Moreover, continuous epitopes can be sequential, if they are only defined by the primary protein sequence, or conformational, if they are associated to secondary structure elements (α-helix, ß-sheet, loops). Discontinuous epitopes are, instead, defined by the tertiary structure of the protein. Furthermore, the expression system (bacteria, yeast, insect cells, mammalian cells), the expression condition used (4), the sensitivity of the recombmant antigen to host proteases (5), and the number of steps involved in the purification procedure may have relevant effects on the yield and on the purity of the recombinant antigen.