Phosphatidylinositol mannosides (PIMs) and their related molecules lipomannan (LM) and lipoarabinomannan (LAM) are important components of the mycobacterial cell wall. These molecules mediate host-pathogen interactions and exhibit immunomodulatory activities. The biosynthesis of these lipoglycans is not fully understood. In this study, we have identified a mycobacterial gene (Rv1500) that is involved in the synthesis of PIMs. We have named this gene pimF. Transposon mutagenesis of pimF of Mycobacterium marinum resulted in multiple phenotypes, including altered colony morphology, disappearance of tetracyl-PIM 7 , and accumulation of tetraacyl-PIM 5 . The syntheses of LAM and LM were also affected. In addition, the pimF mutant exhibited a defect during infection of cultured macrophage cells. Although the mutant was able to replicate and persist within macrophages, the initial cell entry step was inefficient. Transformation of the M. marinum mutant with the pimF homolog of Mycobacterium tuberculosis complemented all of the above mentioned phenotypes. These results provide evidence that PimF is a mannosyltransferase. However, sequence analysis indicates that PimF is distinct from mannosyltransferases involved in the early steps of PIM synthesis. PimF catalyzes the formation of high molecular weight PIMs, which are precursors for the synthesis of LAM and LM. As such, this work marks the first analysis of a mannosyltransferase involved in the later stages of PIM synthesis.The mycobacterial cell wall modulates interactions between the tubercle bacillus and its environment. The cell wall consists of a covalently bound structure, the mycolic acid-arabinogalactan-peptidoglycan complex, and a variety of free lipids that complement the mycolate residues to form an asymmetric bilayer (1-3). Among the cell wall-associated lipids, phosphatidylinositol mannosides (PIMs) 1 and their multiglycosylated counterparts lipomannan (LM) and lipoarabinomannan (LAM), have emerged as major factors in mediating host-pathogen interactions and are presumed to be necessary for the survival and persistence of the pathogen within the host (4). LAM has been implicated in various immunomodulatory effects, including the down-regulation of cell-mediated immunity (5). In addition, the mannose-capped LAM found in the slowly growing mycobacteria, including Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium avium, has been shown to bind cell surface receptors of macrophages and dendritic cells and aid invasion of host cells (6 -8).PIMs are known to mediate the attachment of M. tuberculosis to nonphagocytic cells and have been implicated in the recruitment of natural killer T cells, which affects the granulomatous response (9, 10).Mycobacterial LAMs are lipoglycans composed of three structural components: the membrane anchor, which is a mannosyl-phosphatidyl-myo-inositol; the backbone, which consists of two homopolysaccharides, mannopyranose (Manp) and arabinofuranose (Araf); and the capping motif, which varies among...

Human splicing factors Hprp3p and Hprp4p are associated with the U4/U6 small nuclear ribonucleoprotein particle, which is essential for the assembly of an active spliceosome. Currently, little is known about the specific roles of these factors in splicing. In this study, we characterized the molecular interaction between Hprp3p and Hprp4p. Constructs were created for expression of Hprp3p or its mutants in bacterial or mammalian cells. We showed that antibodies against either Hprp3p or Hprp4p were able to pull-down the Hprp3p-Hprp4p complex formed in Escherichia coli lysates. By co-immunoprecipitation and isothermal titration calorimetry, we demonstrated that purified Hprp3p and its mutants containing the central region, but lacking either the N-terminal 194 amino acids or the C-terminal 240 amino acids, were able to interact with Hprp4p. Conversely, Hprp3p mutants containing only the N-or Cterminal region did not interact with Hprp4p. In addition, by co-immunoprecipitation, we showed that intact Hprp3p and its mutants containing the central region interacted with Hprp4p in HeLa cell nuclear extracts. Primer extension analysis illustrated that the central region of Hprp3p is required to maintain the association of Hprp3p-Hprp4p with U4/U6 small nuclear RNAs, suggesting that this Hprp3p/Hprp4p interaction allows the recruitment of Hprp4p, and perhaps other protein(s), to the U4/U6 small nuclear ribonucleoprotein particle.Pre-mRNA splicing occurs in the spliceosome, a large RNAprotein complex that contains a pre-mRNA, four essential small nuclear ribonucleoprotein (snRNP) 1 particles (U1, U2, U5, and U4/U6), and numerous non-snRNP splicing factors (1-3). Each snRNP particle consists of one (U1, U2, and U5) or two (U4/U6) snRNAs complexed with a set of Sm or Sm-like proteins and several particle-specific proteins (4 -6). These snRNPs recognize conserved sequences of the pre-mRNA and assemble into a catalytically active spliceosome that catalyzes the two cleavage-ligation reactions of pre-mRNA splicing (1,2,7,8). Spliceosome assembly follows an ordered pathway through the formation of several intermediate complexes (2). First, a pre-spliceosome (A complex) is formed once U1 and U2 snRNAs (as part of the U1 and U2 snRNPs) associate with the conserved 5Ј-splice site and the branch point of the intron, respectively (9, 10). Then, U4/U6 snRNP, in which U4 and U6 snRNAs base pair over an extended complementary region, forming a Y-shaped junction, is recruited together with U5 snRNP, presumably via protein/protein interactions, to the pre-spliceosome to form the mature spliceosome (B complex) (11-13).Prior to the first catalytic step of splicing, important conformational rearrangements occur to create a catalytically active spliceosome. For example, U1 snRNA dissociates from the 5Ј-splice site, and the U4/U6 snRNA association is disrupted (14 -16), leaving the U6 snRNA free to base pair with both the U2 snRNA and the 5Ј-splice site (17). The U2 and U6 snRNAs, together with the pre-mRNA, may form the catalytic core of the s...

Rifampin is a front-line antibiotic for the treatment of tuberculosis. Infections caused by rifampin-and multidrug-resistant Mycobacterium tuberculosis strains are difficult to treat and contribute to a poor clinical outcome. Rifampin resistance most often results from mutations in rpoB. However, some drug-resistant strains have rpoB alleles that encode the phenotype for susceptibility. Similarly, non-M. tuberculosis mycobacteria exhibit higher levels of baseline resistance to rifampin, despite the presence of rpoB alleles that encode the phenotype for susceptibility. To identify other genes involved in rifampin resistance, we generated a library of Mycobacterium smegmatis mc 2 155 transposon insertion mutants. Upon screening this library, we identified one mutant that was hypersensitive to rifampin. The transposon insertion was localized to the arr gene, which encodes rifampin ADP ribosyltransferase, an enzyme able to inactivate rifampin. Sequence analysis revealed differences in the arr alleles of M. smegmatis strain mc 2 155 and previously described strain DSM 43756. The arr region of strain mc 2 155 contains a second, partial copy of the arr gene plus a novel insertion sequence, IS1623.Mycobacterial infections, including tuberculosis (TB) and leprosy, are bacterial diseases of global importance. The World Health Organization estimates that the worldwide incidences of TB increased 0.4% in 2001, to 8.5 million new cases (28). Control of TB is complicated by its ease of transmission, difficulties in administering the long-course chemotherapy regimens, and the appearance of strains that are multidrug resistant (MDR), which is defined as resistance to the two front-line anti-TB drugs, isoniazid and rifampin. Rifampin is a broad-spectrum antibiotic that inhibits bacterial DNA-dependent RNA polymerase activity. Resistance to rifampin is most often caused by mutations in rpoB, which encodes the ␤ subunit of RNA polymerase (20,25). In rifampin-resistant clinical isolates of Mycobacterium tuberculosis, an estimated 96% of the rpoB mutations map to an 81-bp region (codons 507 to 533) near the middle of the gene (20). Mutations at codons 531, 526, and 516 are the most common (13). A variety of assays that detect these sequence polymorphisms have been developed (3, 23) and allow the rapid determination of the drug susceptibilities of clinical M. tuberculosis isolates. In addition, an estimated 90% of rifampin-resistant clinical isolates are also isoniazid resistant, such that rifampin resistance is a positive indicator of MDR TB. However, about 4% of rifampin-resistant clinical isolates of M. tuberculosis have no mutations in the 81-bp core region or elsewhere in the rpoB gene (3,8,14,20). In addition, some mycobacteria, particularly atypical mycobacteria, such as Mycobacterium smegmatis, Mycobacterium avium, and Mycobacterium intracellulare, are resistant to rifampin, even though they possess sensitive RNA polymerase (9,14,20). These findings indicate that other genes can contribute to rifampin resistance. To identify s...

BCG vaccines are a family of closely related daughter strains of an attenuated isolate of Mycobacterium bovis derived by in vitro passage from 1908 to 1921. During subsequent laboratory propagation of the vaccine strain until its lyophilization in 1961, BCG Pasteur underwent at least seven further genomic mutations. The impact of these mutations on the properties of the vaccine is currently unknown. One mutation, a glycine-to-aspartic acid substitution in the mmaA3 gene, occurred between 1927 and 1931 and impairs methoxymycolic acid synthesis in BCG strains obtained from the Pasteur Institute after this period. Mycolic acids of the cell wall are classified into three functional groups (alpha-, methoxy-, and ketomycolic acids), and together these lipids form a highly specialized permeability barrier around the bacterium. To explore the impact of methoxymycolic acid production by BCG strains, we complemented the functional gene of mmaA3 into BCG Denmark and tested a number of in vitro and in vivo phenotypes. Surprisingly, restoration of methoxymycolic acids alone had no effect on cell wall permeability, resistance to antibiotics, or growth in cultured macrophages and C57BL/6 mice. Our results demonstrate that the loss of methoxymycolic acid production did not apparently affect the virulence of BCG strains.Bacille Calmette-Guérin (BCG) vaccines are live attenuated strains of Mycobacterium bovis derived by in vitro passage from 1908 to 1921. BCG vaccines are given to millions of infants each year as antituberculosis vaccines, although their capacity to prevent tuberculosis in clinical trials has ranged from 80% protection to no detectable benefit (11). Several hypotheses have been proposed to explain this variable protection, including exposure to environmental mycobacteria (26) and differences between BCG vaccine strains (8).From genomic analyses, it is now known that during in vitro passage, M. bovis lost a genomic region called RD1 (19), which has been shown to contribute to the observed attenuation of virulence of M. bovis BCG strains (16,27). However, complementation of RD1 in the Pasteur strain of BCG did not completely restore pathogenicity in immunocompetent mice (27), suggesting that further mutations contribute to the observed phenotype of BCG strains. Because BCG stocks were propagated for another 40 to 50 years in various vaccine production laboratories, it has been hypothesized that ongoing evolution of BCG in vitro may have resulted in additional attenuation to the detriment of protective efficacy (5). Early reports on BCG suggest a second phase of attenuation in the late 1920s (24), with a decrease in BCG virulence in animal models (15) and reduced persistence of BCG in the mesenteric lymph nodes of vaccinated children (35). These observations are consistent with recent genomic analysis of existing BCG strains that revealed numerous mutations occurring after 1921 (6, 23).Further evidence of evolution of BCG strains followed from studies demonstrating the loss of methoxymycolic acid production in BCG P...