A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins was resolved by 2-DE gel. After ConA ligand blotting, 41 proteins were identified by mass spectrometry as putative glycoproteins with 34 of them new probably mannosylated proteins. These results contribute to the construction of the ConA affinity glycoprotein database of M. tuberculosis, and provide useful information for understanding the biological role of glycoproteins in mycobacteria.

The ζ subunit is a novel natural inhibitor of the α-proteobacterial F1FO-ATPase described originally in Paracoccus denitrificans. To characterize the mechanism by which this subunit inhibits the F1FO nanomotor, the ζ subunit of Paracoccus denitrificans (Pd-ζ) was analyzed by the combination of kinetic, biochemical, bioinformatic, proteomic, and structural approaches. The ζ subunit causes full inhibition of the sulfite-activated PdF1-ATPase with an apparent IC50 of 270 nM by a mechanism independent of the ε subunit. The inhibitory region of the ζ subunit resides in the first 14 N-terminal residues of the protein, which protrude from the 4-α-helix bundle structure of the isolated ζ subunit, as resolved by NMR. Cross-linking experiments show that the ζ subunit interacts with rotor (γ) and stator (α, β) subunits of the F1-ATPase, indicating that the ζ subunit hinders rotation of the central stalk. In addition, a putatively regulatory nucleotide-binding site was found in the ζ subunit by isothermal titration calorimetry. Together, the data show that the ζ subunit controls the rotation of F1FO-ATPase by a mechanism reminiscent of, but different from, those described for mitochondrial IF1 and bacterial ε subunits where the 4-α-helix bundle of ζ seems to work as an anchoring domain that orients the N-terminal inhibitory domain to hinder rotation of the central stalk.

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Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.

The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F(1)F(O)-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.