Mycobacterium tuberculosis is the infectious agent giving rise to human tuberculosis. The entire genome of M. tuberculosis, comprising approximately 4000 open reading frames, has been sequenced. The huge amount of information released from this project has facilitated proteome analysis of M. tuberculosis. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to fractions derived from M. tuberculosis culture filtrate, cell wall, and cytosol, resulting in the resolution of 376, 413, and 395 spots, respectively, in silver-stained gels. By microsequencing and immunodetection, 38 culture filtrate proteins were identified and mapped, of which 12 were identified for the first time. In the same manner, 23 cell wall proteins and 19 cytosol proteins were identified and mapped, with 9 and 10, respectively, being novel proteins. One of the novel proteins was not predicted in the genome project, and for four of the identified proteins alternative start codons were suggested. Fourteen of the culture filtrate proteins were proposed to possess signal sequences. Seven of these proteins were microsequenced and the N-terminal sequences obtained confirmed the prediction. The data presented here are an important complement to the genetic information, and the established 2-D PAGE maps (also available at: www.ssi.dk/publichealth/tbimmun) provide a basis for comparative studies of protein expression.

Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor (

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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-c production at levels comparable to M. bovis Bacillus Calmette-Gu erin-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-c 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-c production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Due to the widespread resistance of bacteria to the available drugs, the discovery of new classes of antibiotics is urgently needed, and naturally occurring antimicrobial peptides (AMPs) are considered promising candidates for future therapeutic use. Amphibian skin is one of the richest sources of such AMPs. In the present study we compared the in vitro bactericidal activities of five AMPs from three different species of anurans against multidrug-resistant clinical isolates belonging to species often involved in nosocomial infections (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii). The peptides tested were temporins A, B, and G from Rana temporaria; the fragment from positions 1 to 18 of esculentin 1b [Esc(1-18)] from Rana esculenta; and bombinin H2 from Bombina variegata. When they were tested in buffer, all the peptides were bactericidal against all bacterial species tested (three strains of each species) at concentrations ranging from 0.5 to 48 ⌴, with only a few exceptions. The temporins were found to be more active against gram-positive bacteria, especially when they were assayed in human serum; Esc(1-18) showed fast and strong bactericidal activity, within 2 to 20 min, especially against the gram-negative species, which were killed by Esc(1-18) at concentrations ranging from 0.5 to 1 ⌴; bombinin H2 displayed similar bactericidal activity toward all isolates. Interestingly, while the activities of the temporins and bombinin H2 were almost completely inhibited in the presence of 20% human serum, the activity of Esc(1-18) against the gram-negative species was partially preserved in the presence of 40% serum. This property renders this peptide an attractive molecule for use in the development of new compounds for the treatment of infectious diseases.