Recombinant Mycobacterium smegmatis vaccine candidates.

Mycobacterium smegmatis is a species of rapidly growing saprophytes with a number of properties that make it an effective vaccine vector. Recombinant M. smegmatis expressing protective antigens of different pathogens and molecules modulating the immune responses offers some potential for reduction of the burden of tuberculosis, HIV and hepatitis B infections. This paper discusses the molecular methods used to generate recombinant M. smegmatis and the results obtained with some of these recombinants.

Production and purification of low calcium response protein H of Chlamydophila pneumoniae.

Chlamydophila pneumoniae possesses a type III secretion system (TTSS), which allows the bacteria to secrete effector molecules into the inclusion membrane and into the cytosol of the host cell. Low calcium response protein H (LcrH), as a part of the TTSS, is a chaperone protein expressed from the middle to late stages of the chlamydial developmental cycle. Gene of LcrH (CPn0811) in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin. The purity was checked with mass spectrometry. The samples were used for immunization of BALB/c mice. The inducible E. coli clone, which over-expresses the chlamydial LcrH, permits the study of the biological properties of this protein.

Rv0802c acetyltransferase from Mycobacterium tuberculosis H37Rv.

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.

Cloning, expression and purification of SmpB from Mycobacterium tuberculosis.

SmpB, a small tmRNA binding protein, is essential for trans-translation. 6His and FLAG tagged SmpB was cloned from Mycobacterium tuberculosis H37Rv. It was expressed in Escherichia coli using the T7 promoter-polymerase system. Anti-FLAG M2 agarose was used for its purification. Mycobacterial SmpB copurifies with other proteins. We identified elongation factor EF-Tu in the purified SmpB preparations.

Different staphylococcal strains elicit different levels of production of T-helper 1-inducing cytokines.

Cytokine production has been implicated in the pathogenic mechanisms of infections caused by the staphylococci, since these bacteria may act as strong cytokine inducers. To gain deeper insight into the Th1 immune response activated by these bacteria, we have analyzed the interferon (IFN), interleukin-12 (IL-12) and IL-18-inducing activities of different Staphylococcus aureus (S. aureus), S. epidermidis and S. saprophyticus strains in human monocytes and murine bone marrow macrophages. A large majority of the S. aureus strains elicited the simultaneous production of IL-12 p70 and IFN-alpha in the human monocytes, while the S. epidermidis and S. saprophyticus strains induced only a low level of production, if any, of these cytokines. Furthermore, a majority of the S. aureus strains induced significantly higher IL-12 p70 and IL-18 titers in the murine bone marrow macrophages than did the S. epidermidis and S. saprophyticus strains. As IL-12, IL-18 and IFN-alpha stimulate Th1 differentiation synergistically, we suggest that S. aureus strains bias the immune response toward a Th1 phenotype, whereas S. epidermidis and S. saprophyticus strains provide a weaker stimulus for the production of Th1-inducing cytokines, and accordingly possibly elicit a less extensive Th1-associated adaptive immunity.

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase.

Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis.

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.

The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly.

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.

Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.

The entire rne gene: the remaining questions.

A 30-fold overexpression of the entire rne gene results in a 3-fold increase in RNase E activity. The overexpression of cloned rne fragments suggests that synthesis of the Rne protein is autoregulated and this regulation is related to the RNase E activity of the gene product. The enzyme produced by the cloned gene has an enhanced thermolability. The thermal stability of the temperature-sensitive enzyme increases when the cells are cultivated in the presence of chloramphenicol, which suggests posttranslational alterations.

RNA processing in prokaryotic cells.

RNA processing in Escherichia coli and some of its phages is reviewed here, with primary emphasis on rRNA and tRNA processing. Three enzymes, RNase III, RNase E and RNase P are responsible for most of the primary endonucleolytic RNA processing events. The first two are proteins, while RNase P is a ribozyme. These three enzymes have unique functions and in their absence, the cleavage events they catalyze are not performed. On the other hand a relatively large number of exonucleases participate in the trimming of the 3' ends of tRNA precursor molecules and they can substitute for each other. Primary processing is the first event that happens to the nascent RNA molecule, while in secondary RNA processing, the substrate is a product of a primary processing event. Although most RNA processing occurs in RNP particles, it seems that only in secondary RNA processing is the RNP particle required for the reaction. Bacteria and especially bacteriophages contain self-splicing introns which in cases were probably acquired from other species.

The rne gene and ribonuclease E.

The cloned rne+ gene complements temperature sensitive RNase E mutations and directs the synthesis of a polypeptide. In vitro the RNA transcribed from the rne gene directs the synthesis of a number of polypeptides, one of which is identical in size to the in vivo product of the rne gene. A rabbit reticulocyte cell free extract programmed with this RNA produced RNase E activity. Thus, it is evident that the rne gene is the structural gene for RNase E. However, the in vivo product of the cloned RNase E gene is more thermolabile than the chromosomal gene product. When cells containing the rne plasmid were treated with chloramphenicol, the pre-existing RNase E became less heat labile with time. This leads to the suggestion that in the cell RNase E undergoes post-translational modification(s).

Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell.

Cells overexpressing the RNA-processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm. The RNA-processing enzymes were associated with the membrane fraction. The membrane was further separated to inner and outer membrane and the three RNA-processing enzymes were found in the inner membrane fraction. By assaying for these enzymatic activities we showed that even in a normal wild-type strain of Escherichia coli these enzymes fractionate primarily with the membrane. The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity. The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed.

The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene.

A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.

Sequencing and expression of the rne gene of Escherichia coli.

RNase E is a major endonucleolytic RNA processing enzyme in Escherichia coli. We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame. Upstream from the gene, there are appropriate consensus sequences for a putative promoter and a ribosome binding site. We have translated this gene using a T7 RNA polymerase/promoter system. We determined 25 amino acids from the N-terminal of the translated product and they are in full agreement with the DNA sequence. The translated product of the rne gene migrates in SDS containing polyacrylamide gels as a 110,000 Da polypeptide, but the open reading frame found in the sequenced DNA indicates a much smaller protein. The entity that migrates as a 110,000 Da contains RNA, which could account, at least partially, for the migration of the rne gene product in SDS containing polyacrylamide gels.

Maturation of precursor 10Sa RNA in Escherichia coli is a two-step process: the first reaction is catalyzed by RNase III in presence of Mn2+.

A precursor to 10Sa RNA accumulates in an rne mutant. However, the present studies indicate that RNase III is the enzyme that processes this RNA. Cell extracts prepared from an rne mutant failed to cleave p10Sa RNA, whereas E coli wild type, rne and rnp cell extracts processed p10Sa RNA under specific assay conditions that require the presence of Mn2+ but not under the customary conditions used for assaying RNase III. That the p10Sa cleaving activity is solely RNase III was confirmed by comparing the increase in p10Sa and poly(A).poly(U) cleaving activities in a strain harboring a plasmid carrying an RNase III gene as compared to a normal E coli strain. It is of interest that these 2 substrates are cleaved by RNase III efficiently, but under 2 different assay conditions. In all strains tested, with normal or elevated levels of RNase III, RNase III fractionates predominantly with the membrane. Further characterization of the maturation of 10Sa RNA revealed that the processing of 10Sa RNA is a 2 step reaction involving 2 separate activities, both sensitive to heat and proteinase K treatment. The first step is catalyzed by RNase III, and results in the formation of a molecule, p10Sa', which is larger than the mature 10Sa RNA. The second activity catalyzes the conversion of p10S' to 10Sa RNA, and this step does not require a divalent cation. The second activity is not any of the known processing endoribonucleases, RNase III, E or P, but could be a new enzyme having no obligate requirement for a divalent cation.

Granulocyte-specific monoclonal antibody inhibiting cytotoxicity reactions in the chicken.

Monoclonal antibody (MAb) 1C3, specific for chicken granulocytes, is described for the first time. Treatment of peripheral blood leukocytes with this MAb markedly decreased natural cytotoxicity reaction against the target cell line. The antibody-dependent cellular cytotoxicity (ADCC) activity of purified granulocytes was also severely affected by treatment with MAb 1C3. These results suggest that 1C3 detects a functional surface antigen on chicken granulocytes and support the hypothesis that granulocytes are the main effector cells in natural cytotoxicity in the chicken.

RNA processing: new mutants that affect endonucleolytic processing of RNA.

A strain of Escherichia coli carrying the rne-3071 mutation that affects the RNA processing enzyme ribonuclease E, was mutagenized, and double mutants deficient in RNA processing were isolated. The isolation was based on the appearance of a particular RNA precursor molecule upon infection of an rne mutant with a specific bacteriophage T4 deletion strain. From one of the double mutants the rne mutation was removed, and the new single mutant, designated rng, was examined. In this mutant the maturation of host RNA as well as of bacteriophage T4 RNA is affected. The effect of the rng mutation on RNA synthesis is unique and can be distinguished from the effects of the other established mutations in RNA processing. The effects of the rng mutation can be recognized in vivo and in vitro.