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Macrophages infected with Mycobacterium tuberculosis undergo increased rates of apoptosis. Important objectives are to define the microbial factors that cause apoptosis, the mechanisms involved and the impact on infection. The 19-kDa M. tuberculosis glycolipoprotein (p19) is both cell wall-associated and secreted and is a candidate virulence factor. We investigated the potential of recombinant, His-tagged p19 lacking the secretion/acylation signal to induce macrophage apoptosis. The TUNEL assay and annexin V binding to membrane phosphatidylserine were used to measure apoptosis. The results show that p19 does act to induce apoptosis in differentiated THP-1 cells and monocyte-derived macrophages and that this effect is both dose- and time-dependent. Furthermore, this effect of p19 is Toll-like receptor (TLR)-2-mediated because preincubation of either THP-1 cells or TLR-2-expressing CHO cells with anti-TLR-2 mAb inhibited apoptosis induced by p19. Apoptosis of macrophages in response to p19 was found to be caspase-8 dependent and caspase-9 independent consistent with a transmembrane pathway signaling cell death through TLR-2. The viability of M. tuberculosis in cells undergoing apoptosis induced by p19 was significantly reduced suggesting the possibility that this may favor containment of infection. Although native p19 is a mycobacterial glycolipoprotein, based upon the use of recombinant p19 where the acylation signal had been removed, we conclude that it is the polypeptide component of p19 that is responsible for signaling through TLR-2 and that the lipid moiety is not required.

Synthetic peptides derived from a 45-kDa glycoprotein antigen of Mycobacterium tuberculosis were shown to function as glycosyltransferase acceptors for mannose residues in a mannosyltransferase cell-free assay. The mannosyltransferase activity was localized within both isolated membranes and a P60 cell wall fraction prepared from the rapidly growing mycobacterial strain, Mycobacterium smegmatis. Incorporation of radiolabel from GDP-[(14)C]mannose was inhibited by the addition of amphomycin, indicating that the glycosyl donor for the peptide acceptors was a member of the mycobacterial polyprenol-P-mannose (PPM) family of activated glycosyl donors. Furthermore, a direct demonstration of transfer from the in situ generated PP[(14)C]Ms was also demonstrated. It was also found that the enzyme activity was sensitive to changes in overall peptide length and amino acid composition. Because glycoproteins are present on the mycobacterial cell surface and are available for interaction with host cells during infection, protein glycosyltransferases may provide novel drug targets. The development of a cell-free mannosyltransferase assay will now facilitate the cloning and biochemical characterisation of the relevant enzymes from M. tuberculosis.

Publisher Summary Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Recent years have seen the resurgence of TB, particularly where there is a high prevalence of human immunodeficiency virus (HIV) infection. Most mycobacteria are saprophytes in soil or water and are harmless to humans; however, at some point thousands of years ago, M. tuberculosis became a significant human pathogen. The origins of tuberculosis are both ancient and obscure. Mycobacteria are aerobic to microaerophilic Gram-positive members of the actinomycete family. M. tuberculosis occurs as straight rods which, unlike other members of the actinomycete group, produce neither aerial hyphae nor spores. A marked characteristic of mycobacteria is that they behave anomalously during staining with arylmethane dyes. Recent developments have, however, limited the value of sputum-based AFB staining methods. The rise in the numbers of HIV-infected people in particular has posed significant problems. Many of those who are HIV-infected may become co-infected with normally non-pathogenic mycobacteria such as Mycobacterium avium (M. avium) or they may develop extra pulmonary tuberculosis for which sputum smears would be unhelpful.

Streptomyces longisporoflavus produces the polyketide-polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug. A genomic library of S. longisporoflavus DNA was cloned in Streptomyces lividans and screened to identify tetronasin-resistance determinants. The inclusion of 0.2M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen. Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified. The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus. The tnrB determinant was further localized, by subcloning, to a 2.8 kb KpnI fragment. DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3). The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP-binding domains of the ABC (ATP binding cassette) superfamily of transport-related proteins. The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter. The mechanism of tetronasin resistance mediated by tnrB is probably an ATP-dependent efflux system.

Tetronasin (formerly ICI139603) [l] is a polyether polyketide antibiotic produced during the aerobic growth of the Gram-positive soil organism Streptomyces longisporofluvus. It has been used c o m i a l l y as a cattle food additive where it has been found to improve food utilisation [2] and to combat the onset of acid lactosis [3]. It may well also prove to be commercially viable as an anticwidant for use in the poultry industry.

Four cases were diagnosed as schizophrenic by careful observation and documentation. The patients had the following in common: 1. By all symptoms no schizophrenia was immediately apparent. It required time and attention to establish the diagnosis in all cases. But then it remained firmly established by the basic accepted criteria. 2. The patients were persons of good intelligence and had other assets as well. They tended to mobilize many mechanisms of defense as "ego" structure threatened to "weaken" further. These consisted both of additional neurotic defenses and some tendency toward "acting-out," but no change was manifested in areas considered schizophrenic. Therefore, we are considering a category of individuals who belong to the group of schizophrenias according to legitimate definition, but apparently no more capable of disorganization, or display of blatant "accessory" symptoms, than the next person. In each of these instances, schizophrenic psychopathology comprised one part of the total picture,...