SummaryMycobacterium tuberculosis infects one-third of the world's population and causes two million deaths annually. The unusually low permeability of its cell wall contributes to the ability of M. tuberculosis to grow within host macrophages, a property required for pathogenesis of infection. Mycobacterium marinum is an established model for discovering genes involved in mycobacterial infection. Mycobacterium marinum mutants with transposon insertions in the b b b bketoacyl-acyl carrier protein synthase B gene ( kasB ) grew poorly in macrophages, although growth in vitro was unaffected. Detailed analyses by thin-layer chromatography, nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-offlight mass spectrometry, infrared spectroscopy, and chemical degradations showed that the kasB mutants synthesize mycolic acids that are 2-4 carbons shorter than wild type; the defect was localized to the proximal portion of the meromycolate chain. In addition, these mutants showed a significant ( ~ 30%) reduction in the abundance of keto-mycolates, with a slight compensatory increase of both a a a a -and methoxymycolates. Despite these small changes in mycolate length and composition, the kasB mutants exhibited strikingly altered cell wall permeability, leading to a marked increase in susceptibility to lipophilic antibiotics and the host antimicrobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were fully complemented by expressing M. tuberculosis kasB , but not by the closely related gene kasA . These studies identify kasB as a novel target for therapeutic intervention in mycobacterial diseases.

Human immunodeficiency virus (HIV)-infected patients respond poorly to hepatitis B vaccination. Records of 194 HIV-infected patients were reviewed for factors associated with successful hepatitis B vaccination. Thirty-four patients (17.5%) developed a protective antibody response. In a logistic regression model, only a plasma HIV RNA level of <400 copies/mL at the time of vaccination was associated with a protective antibody response (P=.003).

Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood. Mycobacterium marinum has recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship to Mycobacterium tuberculosis and because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of the M. marinum model, we have developed efficient transposon mutagenesis of the organism by using a Drosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous to Rv2603c and Rv2604c of M. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation with M. tuberculosis genomic DNA encompassing Rv2603c to Rv2606c corrected the pigmentation and growth defects of the mutant. These data demonstrate the utility of mariner-based transposon mutagenesis of M. marinum and that M. marinum can be used to study the function of M. tuberculosis genes involved in intracellular survival and replication.

The attachment of mycobacteria to fibronectin is well documented (1, 2). All tested species including Mycobacterium bovis strain BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium leprae, and Mycobacterium smegmatis were observed to attach to fibronectin (3-5). In studies on M. bovis BCG-mediated immunotherapy, which is the treatment of choice for superficial bladder cancer, fibronectin attachment was shown to be necessary for the expression of antitumor activity (2). In addition, the attachment and internalization of M. bovis BCG, M. avium, and M. leprae by epithelial cells and Schwann cells also were shown to be dependent on bacterial attachment to fibronectin (4, 6, 7). Thus, an understanding of the interaction between mycobacterial proteins mediating attachment to fibronectin is needed.Two distinct mycobacterial proteins or protein complexes, fibronectin attachment protein (FAP) 1 and proteins of the antigen 85 complex, have been linked to mycobacterial attachment to fibronectin (8, 9). The interaction of proteins from the antigen 85 complex with fibronectin has been characterized using recombinant 85A, 85B, and 85C proteins (10 -12). The best characterized of the complex is the 85B protein. In the present study, the amino acids required for fibronectin binding of the FAP-A-(269 -292) peptide were determined us-

There is no difference in the frequency or prognostic significance of viral blips between patients receiving NNRTI-based therapy and patients receiving PI-based therapy. Our results suggest that viral blips occur at a similar rate among patients receiving NNRTI-based regimens and patients receiving PI-based regimens and that they are not predictive of virologic failure.