Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Gué rin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting ⌬RD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis ⌬RD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the ⌬RD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Gué rin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness. B acillus Calmette-Guérin (BCG) was first isolated fromMycobacterium bovis after serial subculturing in ox bile medium (1, 2), when Drs. Calmette and Guérin set out to test the hypothesis that a bovine tubercle bacillus could transmit pulmonary tuberculosis after oral administration (1, 3, 4). However, unexpectedly after the 39th passage, the strain was unable to kill experimental animals (1, 2), and showed no reversion to virulence even after the authors had performed over 200 passages (3), which is consistent with the attenuating mutation being a deletion mutation. In proceeding studies, BCG was determined to be able to protect animals receiving a lethal challenge of virulent tubercle bacilli (5), and in 1921 was first used as an anti-tuberculous vaccine (6). Presently, an estimated 3 billion doses have been used to vaccinate the human population against tuberculosis, yet the mechanism that causes the attenuation of BCG remains unknown.Mahairas et al. (6) first compared the genomic sequences of BCG and M. bovis, by using subtractive hybridization, and found that there were three regions of difference (designated RD1, RD2, and RD3) present in the genome of M. bovis, but missing in BCG. Behr et al. (7), and others (8), later identified 16 large deletions, including RD1-RD3, which were present in the Mycobacterium tuberculosis genome but absent in BCG. Eleven of these 16 deletions were unique to M. bovis whereas the remaining 5 del...

Host-pathogen interactions are often driven by mechanisms that promote genetic variability. We have identified a group of temperate bacteriophages that generate diversity in a gene, designated mtd (major tropism determinant), which specifies tropism for receptor molecules on host Bordetella species. Tropism switching is the result of a template-dependent, reverse transcriptase-mediated process that introduces nucleotide substitutions at defined locations within mtd. This cassette-based mechanism is capable of providing a vast repertoire of potential ligand-receptor interactions.

Amyloid or amyloid-like fibrils are elongated, insoluble protein aggregates, formed in vivo in association with neurodegenerative diseases or in vitro from soluble native proteins, respectively. The underlying structure of the fibrillar or 'cross-beta' state has presented long-standing, fundamental puzzles of protein structure. These include whether fibril-forming proteins have two structurally distinct stable states, native and fibrillar, and whether all or only part of the native protein refolds as it converts to the fibrillar state. Here we show that a designed amyloid-like fibril of the well-characterized enzyme RNase A contains native-like molecules capable of enzymatic activity. In addition, these functional molecular units are formed from a core RNase A domain and a swapped complementary domain. These findings are consistent with the zipper-spine model in which a cross-beta spine is decorated with three-dimensional domain-swapped functional units, retaining native-like structure.

The postsynaptic density (PSD) is a complex assembly of proteins associated with the postsynaptic membrane that organizes neurotransmitter receptors, signaling pathways, and regulatory elements within a cytoskeletal matrix. Here we show that the sterile alpha motif domain of rat Shank3/ProSAP2, a master scaffolding protein located deep within the PSD, can form large sheets composed of helical fibers stacked side by side. Zn2+, which is found in high concentrations in the PSD, binds tightly to Shank3 and may regulate assembly. Sheets of the Shank protein could form a platform for the construction of the PSD complex.

In humans suffering from dialysis-related amyloidosis, the protein ␤2-microglobulin (␤2M) is deposited as an amyloid; however, an amyloid of ␤2M is unknown in mice. ␤2M sequences from human and mouse are 70% identical, but there is a seven-residue peptide in which six residues differ. This peptide from human ␤2M forms amyloid in vitro, whereas the mouse peptide does not. Substitution of the human peptide for its counterpart in the mouse sequence results in the formation of amyloid in vitro. These results show that a seven-residue segment of human ␤2M is sufficient to convert ␤2M to the amyloid state, and that specific residue interactions are crucial to the conversion. These observations are consistent with a proposed Zipper-spine model for ␤2M amyloid, in which the spine of the fibril consists of an anhydrous ␤-sheet. More than 20 proteins have been found to aggregate into amyloids, elongated unbranched fibrils that bind the aromatic dyes Congo red and ThioflavinT (ThT) and have a common cross ␤ x-ray diffraction pattern (1, 2). The proteins that form amyloids differ in size, function, sequence, and native structure, but all form aggregates similar in structure and properties (3-5). It has long been recognized from the cross-␤ diffraction pattern that amyloids are formed from ␤-sheets Ϸ10-12 Å apart, each made up of extended strands stacked Ϸ4.7 Å apart (6, 7). There is evidence that in some amyloids, the ␤-strands run parallel to each other (8-10), and in others they may run antiparallel (11,12).Some models for amyloid structure depict the entire native protein as refolding into the amyloid (13-16); we term these Entire-refolding models. Other models depict the interactions of amyloid to be formed from only a small segment of the protein, with the rest retaining a native-like structure (17)(18)(19)(20). Entirerefolding models are based in part on the idea that amyloid formation is an inexorable tendency of all proteins, and that variations in rate of achieving the amyloid state are mainly a matter of amino acid composition (21). In contrast, models that depict amyloid formation as having its basis in a ''gain of interaction'' (18) focus on the formation of a new intermolecular bond contributed by a segment of the entire protein. The formation of these intermolecular bonds would in principle depend on the amino acid sequence, not just the composition. In this paper, we focus on a particular gain-of-interaction model, called the Zipper-spine model, in which the new interaction is a spine of ␤-sheet (17).One of the most intensively studied amyloid-forming proteins is ␤2-microglobulin (␤2M), a normally soluble protein that aggregates into pathogenic fibrils either at low pH (22) or under physiological conditions when divalent copper is present (23). The Entire-refolding view of amyloid depicts dialysis-related amyloidosis pathogenesis as destabilization of the native structure of ␤2M followed by formation of a nucleating ␤2M species that forms amyloid fibrils (24-26). However, there is accumulating evidence that s...

We present an experimental study of the self-assembly of capsid proteins of the cowpea chlorotic mosaic virus (CCMV), in the absence of the viral genome, as a function of pH and ionic strength. In accord with previous measurements, a wide range of polymorphs can be identified by electron microscopy, among them single and multiwalled shells and tubes. The images are analyzed with respect to size and shape of aggregates, and evidence is given that equilibrium has been achieved, allowing a phase diagram to be constructed. Some previously unreported structures are also described. The range and stability of the polymorphs can be understood in terms of electrostatic interactions and the way they affect the spontaneous curvature of protein networks and the relative stabilities of pentamers and hexamers.

Ferritins are known as important iron storage/detoxification proteins and are widely found in living organisms. This report details the 2.1 A resolution native and 2.7 A resolution iron bound structures of the ferritin from the hyperthermophilic Archaeon Archaeoglobus fulgidus, and represents the first structure of a ferritin from an archaeon, or a hyperthermophilic organism. The A. fulgidus ferritin (AfFtn) monomer has a high degree of structural similarity with archetypal ferritins from E. coli and humans, but the AfFtn quaternary structure is novel; 24 subunits assemble into a shell having tetrahedral (2-3) rather than the canonical octahedral (4-3-2) symmetry of archetypal ferritins. The difference in assembly opens four large (approximately 45 A) pores in the AfFtn shell. Two nonconservative amino acid substitutions may be critical for stabilizing the tetrahedral form.

Amyloid fibrils are associated with several disease states, but their structures have yet to be fully defined. Here we use site-directed spin labeling to explain some of the specific interactions that are formed between subunits when the protein transthyretin (TTR) assembles into amyloid fibrils, which are associated with both spontaneous and familial amyloid diseases in humans. The results suggest that fibrils are formed when a major conformational change displaces the terminal beta-strand from the edge of a beta-sheet in the native structure, exposing the penultimate strand. The newly exposed strand then allows a novel beta-sheet interaction to form between the TTR subunits. This interaction and another previously identified subunit association lead to a plausible model for the specific sequence of beta-strands in one of the indefinitely repeating beta-sheets of TTR amyloid, which is formed by a head-to-head, tail-to-tail arrangement of subunits.